Search

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.

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.

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.

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.

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.

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.

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.

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.

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 (

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 strain is an important indicator of mechanical function, but is difficult to noninvasively measure in the intervertebral disc. The objective of this study was to generate a disc strain template, a 3D average of disc strain, of a group of human L4-L5 discs loaded in axial compression. To do so, magnetic resonance images of uncompressed discs were used to create an average disc shape. Next, the strain tensors were calculated pixel-wise by using a previously developed registration algorithm. Individual disc strain tensor components were then transformed to the template space and averaged to create the disc strain template. The strain template reduced individual variability while highlighting group trends. For example, higher axial and circumferential strains were present in the lateral and posterolateral regions of the disc, which may lead to annular tears. This quantification of group-level trends in local 3D strain is a significant step forward in the study of disc biomechanics. These trends were compared to a finite element model that had been previously validated against the disc-level mechanical response. Depending on the strain component, 81-99% of the regions within the finite element model had calculated strains within one standard deviation of the template strain results. The template creation technique provides a new measurement technique useful for a wide range of studies, including more complex loading conditions, the effect of disc pathologies and degeneration, damage mechanisms, and design and evaluation of treatments. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1264-1273, 2016.

Changes in tendon viscoelastic properties are observed after injuries and during healing as a product of altered composition and structure. Continuous Shear Wave Elastography is a new technique measuring viscoelastic properties of soft tissues using external shear waves. Tendon has not been studied with this technique, therefore, the aims of this study were to establish the range of shear and viscosity moduli in healthy Achilles tendons, determine bilateral differences of these parameters and explore correlations of viscoelasticity to plantar flexion strength and tendon area. Continuous Shear Wave Elastography was performed over the free portion of both Achilles tendons from 29 subjects. Isometric plantar flexion strength and cross sectional area were measured. The average shear and viscous moduli was 83.2kPa and 141.0Pa-s, respectively. No correlations existed between the shear or viscous modulus and area or strength. This indicates that viscoelastic properties can be considered novel, independent biomarkers. The shear and viscosity moduli were bilaterally equivalent (p=0.013,0.017) which allows determining pathologies through side-to-side deviations. The average bilateral coefficient of variation was 7.2% and 9.4% for shear and viscosity modulus, respectively. The viscoelastic properties of the Achilles tendon may provide an unbiased, non-subjective rating system of tendon recovery and optimizing treatment strategies.

Degeneration of the intervertebral discs is strongly implicated as a cause of low back pain. Since current treatments for discogenic low back pain show poor long-term efficacy, a number of new biological strategies are being pursued. For such therapies to succeed, it is critical that they be validated in conditions that mimic the unique biochemical microenvironment of the nucleus pulposus (NP), which include low oxygen tension. Therefore, the objective of this study was to investigate the effects of oxygen tension on NP cell functional extracellular matrix elaboration in 3D culture. Bovine NP cells were encapsulated in agarose constructs and cultured for 14 or 42 days in either 20% or 2% oxygen in defined media containing transforming growth factor beta-3. At each time point, extracellular matrix composition, biomechanics, and mRNA expression of key phenotypic markers were evaluated. Results showed that while bulk mechanics and composition were largely independent of oxygen level, low oxygen promoted improved restoration of the NP phenotype, higher mRNA expression of extracellular matrix and NP specific markers, and more uniform matrix elaboration. These findings indicate that culture under physiological oxygen levels is an important consideration for successful development of cell and growth factor-based regenerative strategies for the disc.

Magnetic resonance imaging (MRI) with T2-weighting is routinely performed to assess intervertebral disc degeneration. Standard clinical evaluations of MR images are qualitative, however, and do not focus on region-specific alterations in the disc. Utilizing a rabbit needle puncture model, T2 mapping was performed on injured discs to develop a quantitative description of the degenerative process following puncture. To do so, an 18G needle was inserted into four discs per rabbit (L3/L4 to L6/L7) and T2 maps were generated pre- and 4 weeks post-injury. Individual T2 maps were normalized to a disc-specific coordinate system and then averaged for pre- and post-injury population composite T2 maps. We also developed a method to automatically segment the nucleus pulposus by fitting the NP region of the T2 maps with modified 2-D and 3-D Gaussian distribution functions. Puncture injury produced alterations in MR signal intensity in a region-specific manner mirroring human degeneration. Population average T2 maps provided a quantitative representation of the injury response, and identified deviations of individual degenerate discs from the pre-injury population. We found that the response to standardized injury was modest at lower lumbar levels, likely as a result of increased disc dimensions. These tools will be valuable for the quantitative characterization of disc degeneration in future clinical and pre-clinical studies. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:140–148, 2015.

Intervertebral disc mechanics are affected by both disc shape and disc degeneration, which in turn each affect the other; disc mechanics additionally have a role in the etiology of disc degeneration. Finite element analysis (FEA) is a favored tool to investigate these relationships, but limited data for intervertebral disc 3D shape has forced the use of simplified or single-subject geometries, with the effect of inter-individual shape variation investigated only in specialized studies. Similarly, most data on disc shape variation with degeneration is based on 2D mid-sagittal images, which incompletely define 3D shape changes. Therefore, the objective of this study was to quantify inter-individual disc shape variation in 3D, classify this variation into independently-occurring modes using a statistical shape model, and identify correlations between disc shape and degeneration. Three-dimensional disc shapes were obtained from MRI of 13 human male cadaver L3L4 discs. An average disc shape and four major modes of shape variation (representing 90% of the variance) were identified. The first mode represented disc axial area and was significantly correlated to degeneration (R(2)=0.44), indicating larger axial area in degenerate discs. Disc height variation occurred in three distinct modes, each also involving non-height variation. The statistical shape model provides an average L3L4 disc shape for FEA that is fully defined in 3D, and makes it convenient to generate a set of shapes with which to represent aggregate inter-individual variation. Degeneration grade-specific shapes can also be generated. To facilitate application, the model is included in this paper׳s supplemental content.

The aim of functional tissue engineering is to repair and replace tissues that have a biomechanical function, i.e., connective orthopaedic tissues. To do this, it is necessary to have accurate benchmarks for the elastic, permeability, and swelling (i.e., biphasic-swelling) properties of native tissues. However, in the case of the intervertebral disc, the biphasic-swelling properties of individual tissues reported in the literature exhibit great variation and even span several orders of magnitude. This variation is probably caused by differences in the testing protocols and the constitutive models used to analyze the data. Therefore, the objective of this study was to measure the human lumbar disc annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous endplates (CEP) biphasic-swelling properties using a consistent experimental protocol and analyses. The testing protocol was composed of a swelling period followed by multiple confined compression ramps. To analyze the confined compression data, the tissues were modeled using a biphasic-swelling model, which augments the standard biphasic model through the addition of a deformation-dependent osmotic pressure term. This model allows considering the swelling deformations and the contribution of osmotic pressure in the analysis of the experimental data. The swelling stretch was not different between the disc regions (AF: 1.28±0.16; NP: 1.73±0.74; CEP: 1.29±0.26), with a total average of 1.42. The aggregate modulus (Ha) of the extra-fibrillar matrix was higher in the CEP (390kPa) compared to the NP (100kPa) or AF (30kPa). The permeability was very different across tissue regions, with the AF permeability (64 E(-16)m(4)/Ns) higher than the NP and CEP (~5.5 E(-16)m(4)/Ns). Additionally, a normalized time-constant (3000s) for the stress relaxation was similar for all the disc tissues. The properties measured in this study are important as benchmarks for tissue engineering and for modeling the disc's mechanical behavior and transport.

The mechanical properties of the human supraspinatus tendon (SST) are highly heterogeneous and may reflect an important adaptive response to its complex, multiaxial loading environment. However, these functional properties are associated with a location-dependent structure and composition that have not been fully elucidated. Therefore, the objective of this study was to determine the concentrations of types I, II and III collagen in six distinct regions of the SST and compare changes in collagen concentration across regions with local changes in mechanical properties. We hypothesized that type I collagen content would be high throughout the tendon, type II collagen would be restricted to regions of compressive loading and type III collagen content would be high in regions associated with damage. We further hypothesized that regions of high type III collagen content would correspond to regions with low tensile modulus and a low degree of collagen alignment. Although type III collagen content was not significantly higher in regions that are frequently damaged, all other hypotheses were supported by our results. In particular, type II collagen content was highest near the insertion while type III collagen was inversely correlated with tendon modulus and collagen alignment. The measured increase in type II collagen under the coracoacromial arch provides evidence of adaptation to compressive loading in the SST. Moreover, the structure-function relationship between type III collagen content and tendon mechanics established in this study demonstrates a mechanism for altered mechanical properties in pathological tendons and provides a guideline for identifying therapeutic targets and pathology-specific biomarkers.

The mechanical behavior of the annulus fibrosus (AF) of the intervertebral disc can be modeled as a mixture of fibers, extra-fibrillar matrix (EFM), ions, and fluid. However, the properties of the EFM have not been measured directly. We measured mechanical properties of the human EFM at several locations, determined the effect of age and degeneration, and evaluated whether changes in EFM properties correspond to AF compositional changes. EFM mechanical properties were measured using a method that combines osmotic loading and confined compression. AF samples were dissected from several locations, and mechanical properties were correlated with age, degeneration, and composition. EFM modulus was found to range between 10 and 50 kPa, increasing nonlinearly with compression magnitude and being highest in the AF outer-anterior region. EFM properties were not correlated with composition or degeneration. However, the EFM modulus, its relative contribution to tissue modulus, and model parameters were correlated with age. These measurements will result in more accurate predictions of deformations in the intervertebral disc. Additionally, parameters such as permeability and diffusivity used for biotransport analysis of glucose and other solutes depend on EFM deformation. Consequently, the accuracy of biotransport simulations will be greatly improved.

Background context Reduced spinal loading degrades the intervertebral disc and alters the muscle size. Purpose To determine the ability of high-frequency and low-intensity vibrations to maintain disc biomechanics and prevent muscle changes during hindlimb unloading. Study design Three groups of Sprague-Dawley rats were hindlimb unloaded for 4 weeks. In two hindlimb unloaded groups, unloading was interrupted for 15 min/d and the rats were positioned upright on a 90 Hz vertically oscillating plate or a sham control inactive plate. One author owns (provisional) patents regarding the application of vibrations to the musculoskeleton. Methods The motion segments L4–L5 were mechanically evaluated in compression-tension, axial creep, and torsion loading. In vivo microcomputed tomography was used to determine longitudinal psoas and paraspinal muscle area. This work was supported by National Institutes of Health, National Aeronautics and Space Administration (NASA), Alliance for Graduate Education and the Professoriate, and NASA-Harriett G. Jenkins Predoctoral and W. Burghardt Turner Fellowships. The author (SJ) holds (provisional) patents regarding the application of vibrations. Results There were no differences between the discs of uninterrupted unloading and sham animals and these groups were pooled. Compared with normally ambulating age-matched controls, hindlimb unloaded discs had altered properties in every loading modality. Psoas area of the unloaded rats increased at L4 and L5 and the paraspinal area decreased at L4. Vibrations (90 Hz) maintained compression-tension properties, partially maintained creep properties, but did not mitigate torsional weakening because of unloading. Low-intensity vibrations prevented the increase in psoas area but did not abate paraspinal muscle loss. Conclusions In support of clinical studies, unloading deconditioned the rodent disc and altered the muscle area. Although brief exposures to upright posture provided only limited benefits, low-intensity vibrations superimposed on upright posture served to preserve disc mechanics during unloading.

Low back pain is a significant socioeconomic burden and intervertebral disc degeneration has been implicated as a cause. A reliable animal model of disc degeneration is necessary to evaluate therapeutics, and functional metrics are essential to quantify their benefit. To this end, needle puncture injuries were created in the caudal intervertebral discs of mice to induce disc degeneration. Compression, torsion, and creep mechanics were assessed both immediately and after eight weeks to distinguish between the effects of injury and the subsequent reparative or degenerative response. Two needle sizes (29 and 26 gauge) were used to determine injury size-dependence. Compressive stiffness (62%), torsional stiffness (60%), and early damping stiffness (84%) decreased immediately after injury with the large needle (26G). These mechanical properties did not change over time despite structural and compositional changes. At 8 weeks following large needle injury, disc height decreased (37%), nucleus pulposus (NP) glycosaminoglycan content decreased (41%), and NP collagen content increased (45%). The small needle size had no significant effect on mechanics and did not initiate degenerative changes in structure and composition. Thus, the injection of therapeutics into the NP with a minimal needle size may limit damage due to the needle insertion. These findings, along with the wide commercial availability of mouse-specific biological probes, indicate that the mouse caudal disc model can be a powerful tool for investigating disc degeneration and therapy.

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

Summary Objective The objective of this study was to establish a large animal model that recapitulates the spectrum of intervertebral disc degeneration that occurs in humans and which is suitable for pre-clinical evaluation of a wide range of experimental therapeutics. Design Degeneration was induced in the lumbar intervertebral discs of large frame goats by either intradiscal injection of chondroitinase ABC (ChABC) over a range of dosages (0.1U, 1U or 5U) or subtotal nucleotomy. Radiographs were used to assess disc height changes over 12 weeks. Degenerative changes to the discs and endplates were assessed via magnetic resonance imaging (MRI), semi-quantitative histological grading, microcomputed tomography (μCT), and measurement of disc biomechanical properties. Results Degenerative changes were observed for all interventions that ranged from mild (0.1U ChABC) to moderate (1U ChABC and nucleotomy) to severe (5U ChABC). All groups showed progressive reductions in disc height over 12 weeks. Histological scores were significantly increased in the 1U and 5U ChABC groups. Reductions in T2 and T1ρ, and increased Pfirrmann grade were observed on MRI. Resorption and remodeling of the cortical boney endplate adjacent to ChABC-injected discs also occurred. Spine segment range of motion (ROM) was greater and compressive modulus was lower in 1U ChABC and nucleotomy discs compared to intact. Conclusions A large animal model of disc degeneration was established that recapitulates the spectrum of structural, compositional and biomechanical features of human disc degeneration. This model may serve as a robust platform for evaluating the efficacy of therapeutics targeted towards varying degrees of disc degeneration.

STUDY DESIGN Investigation of injectable nucleus pulposus (NP) implant. OBJECTIVE To assess the ability of a recently developed injectable hydrogel implant to restore nondegenerative disc mechanics through support of NP functional mechanics. SUMMARY OF BACKGROUND DATA Although surgical intervention for low back pain is effective for some patients, treated discs undergo altered biomechanics and adjacent levels are at increased risk for accelerated degeneration. One potential treatment as an alternative to surgery for degenerated disc includes the percutaneous delivery of agents to support NP functional mechanics. The implants are delivered in a minimally invasive fashion, potentially on an outpatient basis, and do not preclude later surgical options. One of the challenges in designing such implants includes the need to match key NP mechanical behavior and mimic the role of native nondegenerate NP in spinal motion. METHODS The oxidized hyaluronic acid gelatin implant material was prepared. In vitro mechanical testing was performed in mature ovine bone-disc-bone units in 3 stages: intact, discectomy, and implantation versus sham. Tested samples were cut axially for qualitative structural observations. RESULTS Discectomy increased axial range of motion (ROM) significantly compared with intact. Hydrogel implantation reduced ROM 17% (P < 0.05) compared with discectomy and returned ROM to intact levels (ROM intact 0.71 mm, discectomy 0.87 mm, postimplantation 0.72 mm). Although ROM for the hydrogel implant group was statistically unchanged compared with the intact disc, ROM for sham discs, which received a discectomy and no implant, was significantly increased compared with intact. The compression and tension stiffness were decreased with discectomy and remained unchanged for both implant and sham groups as expected because the annulus fibrosus was not repaired. Gross morphology images confirmed no ejection of NP implant. CONCLUSION An injectable implant that mimics nondegenerate NP has the potential to return motion segment ROM to normal subsequent to injury.

Experimental measurement and normalization of in vitro disc torsion mechanics and collagen content for several animal species used in intervertebral disc research and comparing these with the human disc.To aid in the selection of appropriate animal models for disc research by measuring torsional mechanical properties and collagen content.There is lack of data and variability in testing protocols for comparing animal and human disc torsion mechanics and collagen content.Intervertebral disc torsion mechanics were measured and normalized by disc height and polar moment of inertia for 11 disc types in 8 mammalian species: the calf, pig, baboon, goat, sheep, rabbit, rat, and mouse lumbar discs, and cow, rat, and mouse caudal discs. Collagen content was measured and normalized by dry weight for the same discs except the rat and the mouse. Collagen fiber stretch in torsion was calculated using an analytical model.Measured torsion parameters varied by several orders of magnitude across the different species. After geometric normalization, only the sheep and pig discs were statistically different from human discs. Fiber stretch was found to be highly dependent on the assumed initial fiber angle. The collagen content of the discs was similar, especially in the outer annulus where only the calf and goat discs were statistically different from human. Disc collagen content did not correlate with torsion mechanics.Disc torsion mechanics are comparable with human lumbar discs in 9 of 11 disc types after normalization by geometry. The normalized torsion mechanics and collagen content of the multiple animal discs presented are useful for selecting and interpreting results for animal disc models. Structural organization of the fiber angle may explain the differences that were noted between species after geometric normalization.

Study Design. Biomechanics of human intervertebral discs before and after nucleotomy. Objective. To noninvasively quantify the effect of nucleotomy on internal strains under axial compression inexion, neutral, and extension positions, and to determine whether the change in strains depended on degeneration. Summary of Background Data. Herniation and nucleotomy may accelerate the progression of disc degeneration. Removal of nucleus pulposus (NP) tissue has resulted in altered disc mechanics in vitro , including a decrease in internal pressure and an increase in the deformations at physiologically relevant strains. We recently presented a technique to quantify internal disc strains using magnetic resonance imaging (MRI). Methods. Degeneration was quantitatively assessed by the T 1 ρ relaxation time in the NP. Samples were prepared from human levels L3-L4 and/or L4-L5. A 1000-N compressive load was applied while in the magnetic resonance scanner. Nucleotomy was performed by removing 2 g of NP through the posterior-lateral annulusbrosus (AF). The discs were rehydrated, reimaged, and retested. The analyzed parameters include axial deformation, AF radial bulge, and strains. Results. The axial deformation was more compressive after nucleotomy. In the neutral position, the axial deformation after nucleotomy correlated with degeneration (as quantied by T 1 ρ in the NP), with minimal alteration in nondegenerated discs. Nucleotomy altered the radial displacements and strains in the neutral position, such that the inner AF radial bulge decreased and the radial strains were more tensile in the lateral AF and less tensile in the posterior AF. In the bending loading positions the radial strains were not affected by nucleotomy. Conclusion. Nucleotomy alters the internal radial and axial AF strains in the neutral position, which may leave the AF vulnerable to damage and microfractures. In bending, the effects of nucleotomy were minimal, likely due to more of the applied load being directed over the AF. Some of the nucleotomy effects are modulated by degeneration, where the mechanical effect of nucleotomy was magnied in degenerated discs and may further induce mechanical

Due to the inability of current clinical practices to restore function to degenerated intervertebral discs, the arena of disc tissue engineering has received substantial attention in recent years. Despite tremendous growth and progress in this field, translation to clinical implementation has been hindered by a lack of well-defined functional benchmarks. Because successful replacement of the disc is contingent upon replication of some or all of its complex mechanical behaviour, it is critically important that disc mechanics be well characterized in order to establish discrete functional goals for tissue engineering. In this review, the key functional signatures of the intervertebral disc are discussed and used to propose a series of native tissue benchmarks to guide the development of engineered replacement tissues. These benchmarks include measures of mechanical function under tensile, compressive and shear deformations for the disc and its substructures. In some cases, important functional measures are identified that have yet to be measured in the native tissue. Ultimately, native tissue benchmark values are compared to measurements that have been made on engineered disc tissues, identifying measures where functional equivalence was achieved, and others where there remain opportunities for advancement. Several excellent reviews exist regarding disc composition and structure, as well as recent tissue engineering strategies; therefore this review will remain focused on the functional aspects of disc tissue engineering.

BACKGROUND CONTEXT: Vertebral end plate support is necessary for successful lumbar total disc replacement (TDR) surgery. Failure to achieve anterior column support as a result of lumbar TDR device undersizing could lead to implant subsidence and fracture. PURPOSE: The purpose of the study was to examine the compressive biomechanical behavior of the vertebral end plate with varying sizes of disc replacement implants. STUDY DESIGN: The study design comprises a biomechanical investigation using a human cadaveric lumbar spine model. METHODS: Fifty-six vertebrae with intact posterior elements were prepared from 13 fresh frozen lumbar spines. Peripheral quantitative computed tomography was performed to assess regional bone density. Vertebrae were potted and subjected to nondestructive compression testing with a small, medium, and large custom-made implants with the footplate geometry of the ProDisc-LTDR (Synthes Spine, West Chester, PA, USA) system and having no keel. Failure testing was performed using the ProDisc-L implant with an intact keel. Pressure sensor film was used to assess contact pressure and distribution. RESULTS: Therewasalinearcorrelationbetweenpercentcoverageoftheendplateandimplant-end plate stiffness (p5.0001) and an inverse correlation with displacement (p5.01). The difference in implant-end plate stiffness between small-medium, medium-large, and small-large implants was 10.5% (p5.03), 10.2% (p5.02), and 19.6% (p!.0001), respectively. Failure analysis revealed similartrendsforimplantsizing,butonlybonedensitywasfoundtosignificantlycorrelatewithfailure properties (r50.76, p!.0001).Therewas a significant reduction in implant-end plate stiffness of 18% whenthekeelwasintactcomparedtowithoutthekeel(range6‐27%,p5.0008).Pressurefilmanalysis revealed that the implant was loaded peripherally and did not have central contact during nondestructiveloading.Therewasatrendtowardgreatercontactpressurewiththesmallimplantwhencompared with the medium implant (p5.06) and the large implant (p5.06). CONCLUSIONS: Although larger implants reduce end plate displacement, increase apparent implant-end plate stiffness, increase the implant-end plate contact area, and decrease the peak contact pressures, low bone density reduces failure properties. The keel introduces a reduction in stiffness to the implant-end plate interface, the clinical significance of which is currently

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

Tendon exhibits nonlinear stress-strain behavior that may be partly due to movement of collagen fibers through the extracellular matrix. While a few techniques have been developed to evaluate the fiber architecture of other soft tissues, the organizational behavior of tendon under load has not been determined. The supraspinatus tendon (SST) of the rotator cuff is of particular interest for investigation due to its complex mechanical environment and corresponding inhomogeneity. In addition, SST injury occurs frequently with limited success in treatment strategies, illustrating the need for a better understanding of SST properties. Therefore, the objective of this study was to quantitatively evaluate the inhomogeneous tensile mechanical properties, fiber organization, and fiber realignment under load of human SST utilizing a novel polarized light technique. Fiber distributions were found to become more aligned under load, particularly during the low stiffness toe-region, suggesting that fiber realignment may be partly responsible for observed nonlinear behavior. Fiber alignment was found to correlate significantly with mechanical parameters, providing evidence for strong structure-function relationships in tendon. Human SST exhibits complex, inhomogeneous mechanical properties and fiber distributions, perhaps due to its complex loading environment. Surprisingly, histological grade of degeneration did not correlate with mechanical properties.

Background context Rat models with altered loading are used to study disc degeneration and mechano-transduction. Given the prominent role of mechanics in disc function and degeneration, it is critical to measure mechanical behavior to evaluate changes after model interventions. Axial compression mechanics of the rat disc are representative of the human disc when normalized by geometry, and differences between the lumbar and caudal disc have been quantified in axial compression. No study has quantified rat disc torsional mechanics. Purpose Compare the torsional mechanical behavior of rat lumbar and caudal discs, determine the contribution of combined axial load on torsional mechanics, and compare the torsional properties of rat discs to human lumbar discs. Study design Cadaveric biomechanical study. Methods Cyclic torsion without compressive load followed by cyclic torsion with a fixed compressive load was applied to rat lumbar and caudal disc levels. Results The apparent torsional modulus was higher in the lumbar region than in the caudal region: 0.081±0.026 (MPa/°, mean±SD) for lumbar axially loaded; 0.066±0.028 for caudal axially loaded; 0.091±0.033 for lumbar in pure torsion; and 0.056±0.035 for caudal in pure torsion. These values were similar to human disc properties reported in the literature ranging from 0.024 to 0.21MPa/°. Conclusions Use of the caudal disc as a model may be appropriate if the mechanical focus is within the linear region of the loading regime. These results provide support for use of this animal model in basic science studies with respect to torsional mechanics.

STUDY DESIGN Integrating theoretical and experimental approaches for annulus fibrosus (AF) functional tissue engineering. OBJECTIVE Apply a hyperelastic constitutive model to characterize the evolution of engineered AF via scalar model parameters. Validate the model and predict the response of engineered constructs to physiologic loading scenarios. SUMMARY OF BACKGROUND DATA There is need for a tissue engineered replacement for degenerate AF. When evaluating engineered replacements for load-bearing tissues, it is necessary to evaluate mechanical function with respect to the native tissue, including nonlinearity and anisotropy. METHODS Aligned nanofibrous poly-epsilon-caprolactone scaffolds with prescribed fiber angles were seeded with bovine AF cells and analyzed over 8 weeks, using experimental (mechanical testing, biochemistry, histology) and theoretical methods (a hyperelastic fiber-reinforced constitutive model). RESULTS The linear region modulus for phi = 0 degrees constructs increased by approximately 25 MPa, and for phi = 90 degrees by approximately 2 MPa from 1 day to 8 weeks in culture. Infiltration and proliferation of AF cells into the scaffold and abundant deposition of s-GAG and aligned collagen was observed. The constitutive model had excellent fits to experimental data to yield matrix and fiber parameters that increased with time in culture. Correlations were observed between biochemical measures and model parameters. The model was successfully validated and used to simulate time-varying responses of engineered AF under shear and biaxial loading. CONCLUSION AF cells seeded on nanofibrous scaffolds elaborated an organized, anisotropic AF-like extracellular matrix, resulting in improved mechanical properties. A hyperelastic fiber-reinforced constitutive model characterized the functional evolution of engineered AF constructs, and was used to simulate physiologically relevant loading configurations. Model predictions demonstrated that fibers resist shear even when the shearing direction does not coincide with the fiber direction. Further, the model suggested that the native AF fiber architecture is uniquely designed to support shear stresses encountered under multiple loading configurations.

Background: Early diagnosis is a challenge in the treatment of degenerative disc disease. A noninvasive biomarker detecting functional mechanics of the disc is needed. T1ρ-weighted imaging, a spin-lock magnetic resonance imaging technique, has shown promise for meeting this need in in vivo studies demonstrating the clinical feasibility of evaluating both intervertebral discs and articular cartilage. The objectives of the present study were (1) to quantitatively determine the relationship between T1ρ relaxation time and measures of nucleus pulposus mechanics, and (2) to evaluate whether the quantitative relationship of T1ρ relaxation time with the degenerative grade and glycosaminoglycan content extend to more severe degeneration. It was hypothesized that the isometric swelling pressure and compressive modulus would be directly correlated with the T1ρ relaxation time and the apparent permeability would be inversely correlated with the T1ρ relaxation time. Methods: Eight cadaver human lumbar spines were imaged to measure T1ρ relaxation times. The nucleus pulposus tissue from the L1 disc through the S1 disc was tested in confined compression to determine the swelling pressure, compressive modulus, and permeability. The glycosaminoglycan and water contents were measured in adjacent tissue. Linear regression analyses were performed to examine the correlation between the T1ρ relaxation time and the other measured variables. Mechanical properties and biochemical content were evaluated for differences associated with degeneration. Results: A positive linear correlation was observed between the T1ρ relaxation time on the images of the nucleus pulposus and the swelling pressure (r = 0.59), glycosaminoglycan content per dry weight (r = 0.69), glycosaminoglycan per wet weight (r = 0.49), and water content (r = 0.53). No significant correlations were observed between the T1ρ relaxation time and the modulus or permeability. Similarly, the T1ρ relaxation time, swelling pressure, glycosaminoglycan content per dry weight, and water content were significantly altered with degeneration, whereas the modulus and permeability were not. Conclusions: T1ρ-weighted magnetic resonance imaging has a strong potential as a quantitative biomarker of the mechanical function of the nucleus pulposus and of disc degeneration. Clinical Relevance: Several in vivo studies have previously demonstrated the clinical feasibility of using T1ρ-weighted imaging to evaluate both intervertebral discs and articular cartilage. Its application for the diagnosis of disc degeneration looks promising.

Study design Experimental measurement and normalization of in vitro disc axial compression mechanics and glycosaminoglycan and water content for several animal species used in intervertebral disc research. Objective To compare normalized axial mechanical properties and glycosaminoglycan and water content from other species to those of the human disc to aid in selection and interpretation of results in animal disc studies. Summary of background data There is a lack of mechanical and biochemical comparative data from animal intervertebral discs with respect to the human disc. Methods Intervertebral disc axial mechanical properties, glycosaminoglycan, and water content were evaluated for 9 disc types in 7 mammalian species: the calf, pig, baboon, sheep, rabbit, rat and mouse lumbar, and the cow and rat tail. Disc area and height were used for calculation of the normalized mechanical parameters. Glycosaminoglycan content was normalized by dry weight. Results Many directly measured mechanical parameters varied by orders of magnitude. However, these parameters became comparable and often did not show significant differences after geometric normalization. Both glycosaminoglycan and water content revealed similarity across species. Conclusion Disc axial mechanics are very similar across animal species when normalizing by the geometric parameters of disc height and area. This suggests that the disc tissue material properties are largely conserved across animal species. These results provide a reference to compare disc axial mechanics and glycosaminoglycan and water composition of experimental animal models to the human lumbar disc, to aid in both selection and interpretation of experimental disc research.

Study Design. Biomechanical study and literature review. Objectives. To quantify the acute effect of needle diameter on the in vitro mechanical properties of cadaver lumbar discs in the rat and sheep. To review published in vivo animal studies and evaluate disc changes with respect to the relative needle size. Summary of Background Data. There are many cases where a disc needle puncture or injection is applied to animal models: puncture injuries to induce degeneration, chemonucleolysis to induce degeneration, and delivery of disc therapies. It is not clear what role the size of the needle may have in the outcome. Methods. Mechanics were measured after sham phosphate buffered saline injection with a 27 G or 33 G needle in the rat and with a 27 G needle in the sheep. A literature review was performed to evaluate studies in which animal discs were treated with a needle puncture or a sham injection. For each study, the ratio of the needle diameter to disc height (needle:height) was calculated. Results. When the rat was injected with a 27 G needle (52% of disc height), the compression, tension, and neutral zone stiffnesses were 20% to 60% below preinjected values and the neutral zone length was 130% higher; when injected with a 33 G needle (26% of disc height), the only affected property was the neutral zone length, which was only 20% greater. When the sheep was injected with a 27 G needle (10% of disc height), none of the axial properties were different from intact, the torsion stiffness was not different, and the torque range was 15% smaller. Twenty-three in vivo studies in the rat, rabbit, dog, or sheep were reviewed. The disc changes depended on the ratio of needle diameter to disc height as follows: significant changes were not observed for needle:height less than 40%, although between 25% and 40% results were variable and some minor nonsignificant effects were observed, disc changes were universal for needle:height over 40%. Conclusion. A needle puncture may directly alter mechanical properties via nucleus pulposus depressurization and/or anulus fibrosus damage, depending on the relative needle size. As more basic science research is aimed at treating disc degeneration via injection of therapeutic factors, these findings provide guidance in design of animal studies. Such studies should consider the relative needle size and include sham control groups to account for the potential effects of the needle injection.

Finite element (FE) models have become an important tool to study load distribution in the healthy and degenerated disc. However, model predictions require accurate constitutive laws and material properties. As the mechanical properties of the intervertebral disc are regulated by its biochemical composition and fiber-reinforced structure, the relationship between the constitutive behavior of the tissue and its composition requires careful consideration. While numerous studies have investigated the annulus fibrosus compressive and tensile properties, specific conditions required to determine model parameters for the osmoviscoelastic model are unavailable. Therefore, the objectives of this study were (1) to complement the existing material testing in the literature with confined compression and tensile tests on human annulus fibrosus and (2) to use these data, together with existing nucleus pulposus compression data to tune a composition-based, osmoviscoelastic material constitutive law. The osmoviscoelastic material constitutive law and the experimental data were used to describe the fiber and nonfiber properties of the human disc. The compressive material properties of normal disc tissue were Gm = 1.23 MPa, M = 1.57, and = 1.964 ¿ 10-16 m4/Ns; the tensile fiber material parameters were E0 = 77.0 MPa; E = 500 MPa, and = 1.8 ¿ 103 MPa-s. The goodness of fit ranged from 0.88 to 0.96 for the four experimental conditions evaluated. The constitutive law emphasized the interdependency of the strong swelling ability of the tissue and the viscoelastic nature of the collagen fibers. This is especially important for numerical models to further study the load sharing behavior with regard to disc degeneration and regeneration. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Total intervertebral disc replacement with a biologic engineered disc may be an alternative to spinal fusion for treating end-stage disc disease. In previous work, we developed disc-like angle ply structures (DAPS) that replicate the structure and function of the native disc and a rat tail model to evaluate DAPS in vivo. Here, we evaluated a strategy in which, after in vivo implantation, endogenous cells could colonize the acellular DAPS and form an extracellular matrix organized by the DAPS topographical template. To do so, acellular DAPS were implanted into the caudal spines of rats and evaluated over 12 weeks by mechanical testing, histology, and microcomputed tomography. An external fixation device was used to stabilize the implant site and various control groups were included to evaluate the effect of immobilization. There was robust tissue formation within the DAPS after implantation and compressive mechanical properties of the implant matched that of the native motion segment. Immobilization provided a stable site for fibrous tissue formation after either a discectomy or a DAPS implantation, but bony fusion eventually resulted, with segments showing intervertebral bridging after long-term implantation, a process that was accelerated by the implanted DAPS. Thus, while compressive mechanical properties were replicated after DAPS implantation, methods to actively prevent fusion must be developed. Future work will focus on limiting fusion by remobilizing the motion segment after a period of integration, delivering pro-chondrogenic factors, and pre-seeding DAPS with cells prior to implantation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:23-31, 2017.

Surgical treatment for lower back pain related to degenerative disc disease commonly includes discectomy and spinal fusion. While surgical intervention may provide short-term pain relief, it results in altered biomechanics of the spine and may lead to further degenerative changes in adjacent segments. One non-fusion technique currently being investigated is nucleus pulposus (NP) support via either an injectable hydrogel or tissue engineered construct. A major challenge for either approach is to mimic the mechanical properties of native NP. Here we adopt an unconfined compression testing configuration to assess toe-region and linear-region modulus and Poisson's ratio, key functional parameters for NP replacement. Human NP, experimental biocompatible hydrogel formulations composed of hyaluronic acid (HA), PEG-g-chitosan, and gelatin, and conventional alginate and agarose gels were investigated as injectable NP replacements or tissue engineering scaffolds. Testing consisted of a stress-relaxation experiment of 5% strain increments followed by 5-min relaxation periods to a total of 25% strain. Human NP had an average linear-region modulus of 5.39 +/- 2.56 kPa and a Poisson's ratio of 0.62 +/- 0.15. The modulus and Poisson's ratio are important parameters for evaluating the design of implant materials and scaffolds. The synthetic HA-based hydrogels approximated NP well and may serve as suitable NP implant materials.

Despite the critical functions the human cartilage endplate (CEP) plays in the intervertebral disc, little is known about its structural and mechanical properties and their changes with degeneration. Quantifying these changes with degeneration is important for understanding how the CEP contributes to the function and pathology of the disc. Therefore the objectives of this study were to quantify the effect of disc degeneration on human CEP mechanical properties, determine the influence of superior and inferior disc site on mechanics and composition, and simulate the role of collagen fibers in CEP and disc mechanics using a validated finite element model. Confined compression data and biochemical composition data were used in a biphasic-swelling model to calculate compressive extrafibrillar elastic and permeability properties. Tensile properties were obtained by applying published tensile test data to an ellipsoidal fiber distribution. Results showed that with degeneration CEP permeability decreased 50-60% suggesting that transport is inhibited in the degenerate disc. CEP fibers are organized parallel to the vertebrae and nucleus pulposus and may contribute to large shear strains (0.1-0.2) and delamination failure of the CEP commonly seen in herniated disc tissue. Fiber-reinforcement also reduces CEP axial strains thereby enhancing fluid flux by a factor of 1.8. Collectively, these results suggest that the structure and mechanics of the CEP may play critical roles in the solute transport and disc mechanics.

Degenerative disc disease afflicts nearly 12 million people in the United States. Although a single initiating cause of degeneration has not been identified, early degenerative changes occur in the nucleus pulposus.1,2 Breakdown of the large aggregating proteoglycans reduces the capacity of the nucleus pulposus to attract and bind water, leading to a loss of disc hydration and decreased hydrostatic pressure.3,4 Ultimately, degeneration progresses to decreased disc height, structural changes in the lamellar architecture of the anulus fibrosus, anular tears and rim lesions, and the formation of osteophytes.5-7 The success of treatment strategies aimed at halting the progression of disc degeneration will require detection of the early stages of the disease, particularly changes in the extracellular matrix content of the nucleus pulposus. Conventional magnetic resonance imaging (MRI) techniques provide excellent detection of late-stage degenerative changes (i.e., changes in disc morphology, height, hydration, bulge, and herniation).8,9 However, these methods are not sensitive to early degenerative changes in the matrix content of the disc.10 Delayed gadolinium-enhanced MRI has been used to quantify proteoglycan in articular cartilage.11,12 However, the contrast agent must be administered intravenously, and diffusion into the cartilage requires a long time.13 This is a significant limitation in the avascular intervertebral disc, in which the negative fixed charged density of the nucleus pulposus hinders diffusion of ionic contrast agents.14,15 Sodium MRI has also been used to measure proteoglycan in articular cartilage.16-18 However, its clinical use is somewhat limited by a low spatial resolution and the need for instrumentation modifications for use on a clinical scanner. Spin-lock MRI techniques have been used to provide noninvasive measures of degeneration in articular cartilage and may potentially be used to assess degeneration in the intervertebral disc. Spin-lock pulses are low power rf pulses applied directly on-resonance with the Larmor precession frequency, locking the magnetization vector into a rotated frame. The relaxation that occurs after the application of a spin-lock pulse is referred to as spin-lattice relaxation in the rotating frame, or T1ρ relaxation. Spin-lock allows the coupling of spins to frequencies that are generally lower than the Larmor frequency. Therefore, slow motion regimes can be studied, such as low frequency physicochemical interactions between water and extracellular matrix molecules. Thus, matrix changes, such as loss of proteoglycan, will be reflected in the T1ρ parameter. T1ρ-weighting provides T2-like images with the advantage of increased dynamic range to degenerative changes compared to conventional T2-weighting.19 In articular cartilage, T1ρ is strongly correlated with proteoglycan content and, thus, has been shown to detect early osteoarthritic changes.20-22 Recently, T1ρ-weighted images of bovine intervertebral disc tissue have been acquired.23 However, a relationship between T1ρ and intervertebral disc degeneration has not been established, nor has it been shown that T1ρ is sensitive to proteoglycan content in the disc. Thus, the objective of the present study was to demonstrate the use of T1ρ MRI for the assessment of degeneration and proteoglycan content in the human intervertebral disc. Quantitative T1ρ measurements were obtained from cadaveric human intervertebral disc tissue. Degenerative grade was assessed from standard T2-weighted images, and tissue was subsequently analyzed for total sulfated-glycosaminoglycan content, a measure of proteoglycan content.

Diagnostic methods and biomarkers of early disc degeneration are needed as emerging treatment technologies develop (e.g., nucleus replacement, total disc arthroplasty, cell therapy, growth factor therapy) to serve as an alternative to lumbar spine fusion in treatment of low back pain. We have recently demonstrated in cadaveric human discs an MR imaging and analysis technique, spin-lock T(1rho)-weighted MRI, which may provide a quantitative, objective, and non-invasive assessment of disc degeneration. The goal of the present study was to assess the feasibility of using T(1rho) MRI in vivo to detect intervertebral disc degeneration. We evaluated ten asymptomatic 40-60-year-old subjects. Each subject was imaged on a 1.5 T whole-body clinical MR scanner. Mean T(1rho) values from a circular region of interest in the center of the nucleus pulposus were calculated from maps generated from a series of T(1rho)-weighted images. The degenerative grade of each lumbar disc was assessed from conventional T(2)-weighted images according to the Pfirmann classification system. The T(1rho) relaxation correlated significantly with disc degeneration (r=-0.51, P0.01) and the values were consistent with our previous cadaveric study, in which we demonstrated correlation between T(1rho) and proteoglycan content. The technique allows for spatial measurements on a continuous rather than an integer-based scale, minimizes the potential for observer bias, has a greater dynamic range than T(2)-weighted imaging, and can be implemented on a 1.5 T clinical scanner without significant hardware modifications. Thus, there is a strong potential to use T(1rho) in vivo as a non-invasive biomarker of proteoglycan loss and early disc degeneration.

Study design The biphasic compressive material properties of normal and degenerate human nucleus pulposus tissue were measured in confined compression. Objectives The objective of this study was to determine the effects of degeneration and age on the mechanical properties of human nucleus pulposus. Summary of background data The nucleus pulposus exhibits swelling behavior in proportion to proteoglycan content. In shear, the nucleus exhibits both fluid-like and solid-like properties, suggesting a biphasic nature. To date, biphasic compressive properties of human nucleus pulpous have not been reported. Methods Human nucleus pulposus samples were tested in confined compression. Isometric swelling stress and effective aggregate modulus were measured. Linear biphasic theory was used to determine the permeability of the tissue. Mechanical behavior was correlated with proteoglycan and water content. Results Degeneration produced significant decreases in swelling stress (Psw = 0.138 +/- 0.029 MPa nondegenerate, Psw = 0.037 +/- 0.038 MPa degenerate) and effective aggregate modulus (H(A)(eff) = 1.01 +/- 0.43 MPa nondegenerate, H(A)(eff) = 0.44 +/- 0.19 MPa degenerate). Both properties were inversely correlated with proteoglycan content. Permeability increased with degeneration (ka = 0.9 +/- 0.43 x 10(-15) m4/N-s nondegenerate, ka = 1.4 +/- 0.58 x 10(-15) m4/N-s degenerate). Conclusions Swelling is the primary load-bearing mechanism in both nondegenerate and degenerate nucleus pulposus. Knowledge of the biphasic material properties of the nucleus pulposus will aid the development of new treatment strategies for disc degeneration aimed at restoring mechanical function of the intervertebral disc.

The mouse lumbar and tail discs are both used as models to study disc degeneration; however, the mechanical behavior of these two levels has not been compared. The objective of this study was to compare the elastic and viscoelastic mechanical properties of lumbar and tail discs of the mouse under axial compression-tension loading. We hypothesized that tail discs would have a larger transition zone (e.g., neutral zone) and would be less stiff in compression. To test these hypotheses, lumbar and tail bone-disc-bone motion segments were loaded in axial compression and tension. The nonlinear elastic mechanical behavior was examined using a tri-linear curvefit. Elastic behavior of lumbar and tail discs was most different in the low-stiffness transition region (neutral zone), where lumbar discs were nearly twice as stiff over half the axial displacement. In addition, viscoelastic behavior, which was examined using a stretch-exponential curvefit, also showed large lumbar and tail differences, where lumbar discs compressed by 60% of their original height and tail discs by 98% after static creep compression. These results demonstrate that tail discs undergo far more axial displacement than lumbar discs under the same load. These findings are relevant to rodent tail models where chronic loads are applied in vivo to study mechanical pathways of degeneration. Furthermore, the tri-linear model, used here to curvefit the nonlinear compression-tension data, quantified stiffness in the transition zone for the first time, which may prove useful in future disc mechanical studies.

A transversely isotropic biphasic mixture model was applied to tendon in uniaxial tension. Parametric analyses were performed and the sensitivity in predicting material parameters was evaluated. Our results provide quantitative evidence for fluid flow as a mechanism that contributes to tendon viscoelasticity. Transversely isotropic material properties were calculated for mouse tail tendon fascicles. The average transverse modulus (E1) was 0.046 MPa, the fiber-aligned Poisson's ratio (ν31) was 2.73, and the transverse Poisson's ratio (ν21) was 0.96; these properties were not strain-dependent. The fiber-aligned modulus (E3) was strain-dependent and was 20.7 MPa in the toe region and 86.1 MPa in the linear region. These solid matrix properties were consistent with previously published tendon tissue and fascicle data. The fascicle permeability was strain-dependent and was 5.5×10−18 m4/N s in the toe region and 0.32×10−18 m4/N s in the linear region, similar to previously reported meniscus permeability in tension. The similar permeabilities of both fascicle and tissue-level samples suggest that fluid flow from individual fascicles, not the packing of multiple fascicles together, may be the primary barrier to fluid flow in tendon and thus the primary mechanism for viscoelasticity.

Study design Measure the mechanical properties of the mouse and rat disc in compression and torsion. Objectives Validate mouse and rat disc as a biomechanical model of the human disc by comparing the normalized properties in compression and torsion loading. Summary of background data Rodents have been widely used as models to study disc degeneration; however, mechanical assessments of the rodent disc have been limited. Mouse and rat disc mechanical properties have not been determined. Methods Mechanically test mouse and rat motion segments from both the lumbar and the caudal levels in axial compression and torsion. Normalize the stiffness using disc geometry and compare with human motion segment stiffness taken from the literature. Compare lumbar and caudal levels with each other within each species, and test for correlation between mechanics and body weight. Results The average compression stiffness, normalized by geometry, was 2-4 MPa and compared well with human motion segment stiffness in compression (3-9 MPa). The average torsion stiffness, normalized by disc geometry, was 5-11 MPa and compared well with human motion segment stiffness in torsion (2-9 MPa). Differences between the lumbar and caudal levels were observed. For the caudal tail, no correlation between body weight and any compression property was observed, but for the lumbar spine, some correlations were observed. CONCLUSIONS.: This study provides validation for the mouse and rat disc as a mechanical model of the human disc. Correlations between lumbar spine properties and animal body weight provide support for the use of quadruped animal lumbar spines as mechanical models of the bipedal human spine. The differences between lumbar and tail mechanics need further exploration. These findings are important in light of the extensive use of the rodent in disc studies and the expected future utility of genetically engineered mice.