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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.

Introduction Inflammation plays a key role in the progression of intervertebral disc degeneration, a condition strongly implicated as a cause of lower back pain. The objective of this study was to investigate the therapeutic potential of poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with interleukin-1 receptor antagonist (IL-1ra) for sustained attenuation of interleukin-1 beta (IL-1β) mediated degradative changes in the nucleus pulposus (NP), using an in vitro model. Methods IL-1ra was encapsulated in PLGA microspheres and release kinetics were determined over 35 days. NP agarose constructs were cultured to functional maturity and treated with combinations of IL-1β and media conditioned with IL-1ra released from microspheres at intervals for up to 20 days. Construct mechanical properties, glycosaminoglycan content, nitrite production and mRNA expression of catabolic mediators were compared to properties for untreated constructs using unpaired Student's t-tests. Results IL-1ra release kinetics were characterized by an initial burst release reducing to a linear release over the first 10 days. IL-1ra released from microspheres attenuated the degradative effects of IL-1β as defined by mechanical properties, glycosaminoglycans (GAG) content, nitric oxide production and mRNA expression of inflammatory mediators for 7 days, and continued to limit functional degradation for up to 20 days. Conclusions In this study, we successfully demonstrated that IL-1ra microspheres can attenuate the degradative effects of IL-1β on the NP for extended periods. This therapeutic strategy may be appropriate for treating early-stage, cytokine-mediated disc degeneration. Ongoing studies are focusing on testing IL-1ra microspheres in an in vivo model of disc degeneration, as a prelude to clinical translation.

Mucopolysaccharidosis VII (MPS VII) is due to deficient β-glucuronidase (GUSB) activity, which leads to accumulation of chondroitin, heparan, and dermatan sulfate glycosaminoglycans in various tissues including those of the spine. Associated spine disease can be due to abnormalities in the vertebrae, the intervertebral disks, or other spine tissues. The goal of this study was to determine if neonatal gene therapy could prevent lumbar spine disease in MPS VII dogs. MPS VII dogs were injected intravenously with a retroviral vector (RV) expressing canine GUSB at 2 to 3 days after birth, which resulted in transduction of hepatocytes that secreted GUSB into blood. Expression was stable for up to 11 years, and mean survival was increased from 0.4 years in untreated dogs to 6.1 years in treated dogs. Despite a profound positive clinical effect, 6-month-old RV-treated MPS VII dogs still had hypoplastic ventral epiphyses with reduced calcification in the lumbar spine, which resulted in a reduced stiffness and increased range of motion that were not improved relative to untreated MPS VII dogs. At six to 11 years of age, ventral vertebrae remained hypoplastic in RV-treated MPS VII dogs, and there was desiccation of the nucleus pulposus in some disks. Histochemical staining demonstrated that disks did not have detectable GUSB activity despite high serum GUSB activity, which is likely due to poor diffusion into this relatively avascular structure. Thus, neonatal gene therapy cannot prevent lumbar spine disease in MPS VII dogs, which predicts that enzyme replacement therapy (ERT) will similarly be relatively ineffective even if started at birth.

Mucopolysaccharidosis type VII (MPS VII) is characterized by deficient β-glucuronidase (GUSB) activity, which leads to accumulation of chondroitin, heparan and dermatan sulfate glycosaminoglycans (GAGs), and multisystemic disease. MPS VII patients can develop kypho-scoliotic deformity and spinal cord compression due to disease of intervertebral disks, vertebral bodies, and associated tissues. We have previously demonstrated in MPS VII dogs that intervertebral disks degenerate, vertebral bodies have irregular surfaces, and vertebral body epiphyses have reduced calcification, but the pathophysiological mechanisms underlying these changes are unclear. We hypothesized that some of these manifestations could be due to upregulation of destructive proteases, possibly via the binding of GAGs to Toll-like receptor 4 (TLR4), as has been proposed for other tissues in MPS models. In this study, the annulus fibrosus of the intervertebral disk of 6-month-old MPS VII dogs had cathepsin B and K activities that were 117- and 2-fold normal, respectively, which were associated with elevations in mRNA levels for these cathepsins as well as TLR4. The epiphyses of MPS VII dogs had a marked elevation in mRNA for the cartilage-associated gene collagen II, consistent with a developmental delay in the conversion of the cartilage to bone in this region. The spine obtained at autopsy from a young man with MPS VII exhibited similar increased cartilage in the vertebral bodies adjacent to the end plates, disorganization of the intervertebral disks, and irregular vertebral end plate morphology. These data suggest that the pathogenesis of destructive changes in the spine in MPS VII may involve upregulation of cathepsins. Inhibition of destructive proteases, such as cathepsins, might reduce spine disease in patients with MPS VII or related disorders.

Mechanical signals are essential in regulating cell functions such as viability, differentiation, proliferation, and extracellular matrix (ECM) production in load-bearing tissues. However, the current understanding of how macroscopic tissue level strain is transferred to cells is confounded by the highly variable strain fields that arise within the ECM of both native and cell-seeded nanofibrous scaffolds. Moreover, it is unclear how these transmission mechanisms relate to native load bearing tissues. The current study investigates how applied macroscopic tensile strain is transferred to the intercellular ECM and cell nuclei in meniscus, tendon, single lamellar AF, and MSC-seeded scaffolds. The mean microscopic Lagrangian and principal strains in the loading direction (E 22 and e 2 ) of all native tissues and scaffolds were attenuated from the applied strains by 50% and 30–40% respectively. In aligned scaffold, a significant correlation was observed between the mean nuclear strain and E 22 (r2=0.98), where 100% of E 22 transferred to nuclei. Less pronounced strain attenuation in scaffolds compared to native tissues is likely due to more homogeneous microstructure and lack of ECM. In addition, the presence of pericellular matrix in native tissues, along with dense ECM, may shield and regulate strain transfer from the ECM to the subcellular space.

Intervertebral disc degeneration is a cell-mediated process that results in the disruption of its function. These changes are difficult to overturn; consequently, early diagnosis is key for the success of any treatment. Clinically, disc degeneration is diagnosed by analyzing morphological changes characteristic of moderate to advanced stages of degeneration. Therefore, there is a need of a reliable biomarker to characterize the compositional and structural integrity of the disc in early stages of degeneration. In this study, shear modulus is proposed as a new biomarker for disc degeneration and a non-invasive method to measure this mechanical property is presented.

Introduction: The cartilaginous endplate (CEP) is a thin layer of hyaline cartilage which functions as a mechanical barrier between the nucleus pulposus and the vertebral endplate and as a gateway for nutrient transport to the intervertebral disc. To date, the geometry of the CEP has not been well defined. This study visualizes the three-dimensional morphology of the CEP and quantifies CEP thickness via a semi-automatic analysis technique. Methods: Human lumbar motion segments (n=24) were imaged using an optimized 3D FLASH sequence. MRI data were evaluated in the axial and mid-sagittal imaging planes for CEP area, circumference, anterior-posterior width, lateral width, and thickness. CEP thickness was evaluated at five anterior-posterior locations. Results: There was no significant difference in CEP circumference, area, A-P width, or lateral width with respect to superior/inferior location (p>0.7) or disc level (p>0.6) except between L1L2 and L4L5 (p 0.5). Discussion: This study demonstrates the potential for MRI FLASH imaging coupled with automatic geometric quantification in evaluating the CEP as a potential contributor in the degenerative cascade.

Protein crosslinking and glycosaminoglycan (GAG) depletion are characteristic of disc degeneration. The objective of this study is to analyze the role of these biochemical changes on the mechanics of annulus fibrosus extra-fibrillar matrix (EFM). It was found that GAG depletion decreased ionic contribution, but not the nonionic contribution of the EFM. Additionally, crosslinking increased the stiffness of annulus fibrosus EFM.

The heterogeneous composition and mechanical properties of the supraspinatus tendon offer an opportunity for studying the structure-function relationships of fibrous musculoskeletal connective tissues. Previous uniaxial testing has demonstrated a correlation between the collagen fiber angle distribution and tendon mechanics in response to tensile loading both parallel and transverse to the tendon longitudinal axis. However, the planar mechanics of the supraspinatus tendon may be more appropriately characterized through biaxial tensile testing, which avoids the limitation of nonphysiologic traction-free boundary conditions present during uniaxial testing. Combined with a structural constitutive model, biaxial testing can help identify the specific structural mechanisms underlying the tendon’s two-dimensional mechanical behavior. Therefore, the objective of this study was to evaluate the contribution of collagen fiber organization to the planar tensile mechanics of the human supraspinatus tendon by fitting biaxial tensile data with a structural constitutive model that incorporates a sample-specific angular distribution of nonlinear fibers. Regional samples were tested under several biaxial boundary conditions while simultaneously measuring the collagen fiber orientations via polarized light imaging. The histograms of fiber angles were fit with a von Mises probability distribution and input into a hyperelastic constitutive model incorporating the contributions of the uncrimped fibers. Samples with a wide fiber angle distribution produced greater transverse stresses than more highly aligned samples. The structural model fit the longitudinal stresses well (median R2 ≥ 0.96) and was validated by successfully predicting the stress response to a mechanical protocol not used for parameter estimation. The transverse stresses were fit less well with greater errors observed for less aligned samples. Sensitivity analyses and relatively affine fiber kinematics suggest that these errors are not due to inaccuracies in measuring the collagen fiber organization. More likely, additional strain energy terms representing fiber-fiber interactions are necessary to provide a closer approximation of the transverse stresses. Nevertheless, this approach demonstrated that the longitudinal tensile mechanics of the supraspinatus tendon are primarily dependent on the moduli, crimp, and angular distribution of its collagen fibers. These results add to the existing knowledge of structure-function relationships in fibrous musculoskeletal tissue, which is valuable for understanding the etiology of degenerative disease, developing effective tissue engineering design strategies, and predicting outcomes of tissue repair.

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

Intervertebral disc degeneration is characterized by a cascade of cellular, biochemical and structural changes that may lead to functional impairment and low back pain. Interleukin-1 beta (IL-1β) is strongly implicated in the etiology of disc degeneration, however there is currently no direct evidence linking IL-1β upregulation to downstream biomechanical changes. The objective of this study was to evaluate long-term agarose culture of nucleus pulposus (NP) cells as a potential in vitro model system to investigate this. Bovine NP cells were cultured in agarose for 49 days in a defined medium containing transforming growth factor-beta 3, after which both mechanical properties and composition were evaluated and compared to native NP. The mRNA levels of NP cell markers were compared to those of freshly isolated NP cells. Glycosaminoglycan (GAG) content, aggregate modulus and hydraulic permeability of mature constructs were similar to native NP, and aggrecan and SOX9 mRNA levels were not significantly different from freshly isolated cells. To investigate direct links between IL-1β and biomechanical changes, mature agarose constructs were treated with IL-1β, and effects on biomechanical properties, extracellular matrix composition and mRNA levels were quantified. IL-1β treatment resulted in upregulation of a disintegrin and metalloproteinase with thrombospondin motifs 4, matrix metalloproteinase-13 and inducible nitric oxide sythase, decreased GAG and modulus, and increased permeability. To evaluate the model as a test platform for therapeutic intervention, co-treatment with IL-1β and IL-1 receptor antagonist (IL-1ra) was evaluated. IL-1ra significantly attenuated degradative changes induced by IL-1β. These results suggest that this in vitro model represents a reliable and cost-effective platform for evaluating new therapies for disc degeneration.

Disc injury through trauma, vibration loading, or mechanical overload, and the resulting disc degeneration in response to these insults over time are tremendous problems affecting the active and veteran military population. Current treatment options fail to restore disc structure and mechanical function. Our goal in this proposal is to develop methodologies for the engineering and implanting of a functional biologic disc replacement. Significant progress has been made in the last 12 months towards achieving this goal. We have successfully engineered a concentric annulus fibrosus, the functional properties of which improve with culture time. We have shown the dynamic culture further enhances functional matrix deposition. We have shown that a short period of exposure to transforming at a high dose is equal to or better than long term exposure for stem cells cultured in an engineered nucleus pulposus-like hyaluronan hydrogel. We have developed and validated a minimally invasive surgical technique for implantation of our engineered disc. We have successfully performed in implantation of acellular engineered discs. Finally, we have designed and implemented a novel internal fixation device to enhance retention of the engineered disc and stabilize the joint during healing.

Because differentiation of mesenchymal stem cells (MSCs) is enacted through the integration of soluble signaling factors and physical cues, including substrate architecture and exogenous mechanical stimulation, it is important to understand how micro-patterned biomaterials may be optimized to enhance differentiation for the formation of functional soft tissues. In the present work, macroscopic strain applied to MSCs in an aligned nanofibrous microenvironment elicited cellular and nuclear deformations that varied depending on scaffold orientation. Reorientation of aligned, oriented MSCs corresponded at the microscopic scale with the affine approximation of their deformation based on macroscopic strains. Moreover, deformations at the subcellular scale corresponded with scaffold orientation, with changes in nuclear shape depending on the direction of substrate alignment. Notably, these deformations induced changes in gene expression that were also dependent on scaffold and cell orientations. These findings demonstrate that directional biases in substrate microstructure convey direction-dependent mechanosensitivity to MSCs and provide an experimental framework in which to explore the mechanistic underpinnings of this response.

The human supraspinatus tendon (SST) exhibits strong heterogeneity in fiber alignment and material properties [1,2]. The relationship between fiber angle distribution and material properties has been previously described by a structurally based continuum model [3], which provided new quantitative structure-function relationships to explain the observed SST heterogeneity; however, in some locations and testing directions, the model predictions were not consistent with a continuum assumption [3]. More recent analysis of the change in fiber angle during loading showed that samples with less aligned fibers have less affine kinematics in uniaxial tensile loading [4]. That is, in uniaxial tensile testing, where the transverse edges freely contract, the fiber strain did not match the tissue strain. Because the SST is somewhat transversely constrained by surrounding rotator cuff structures in vivo and has distributed fibers to support multidirectional loading, the freely contracting edges of uniaxial tension may not appropriately constrain the tendon. Therefore, the objective of this study was to evaluate SST stress-strain behavior and affine deformation under biaxial tension. Specifically, if behaving as a continuum, we expected that applying a fixed boundary condition in the transverse direction would produce a higher apparent modulus, a smaller toe-region, and more affine fiber realignment than a free boundary condition.Copyright © 2011 by ASME

Intervertebral disc degeneration is characterized by a progressive cascade of structural, biochemical and biomechanical changes affecting the annulus fibrosus (AF), nucleus pulposus (NP) and end plates (EP). These changes are considered to contribute to the onset of back pain. It has been shown that mechanical properties of the AF and NP change significantly with degeneration [1,2]. Therefore, mechanical properties have the potential to serve as a biomarker for diagnosis of disc degeneration. Currently, disc degeneration is diagnosed based on the detection of structural and compositional changes using MRI, X-ray, discography and other imaging techniques. These methods, however, do not measure directly the mechanical properties of the extracellular matrix of the disc. Magnetic Resonance Elastography (MRE) is a technique that has been used to measure in vivo mechanical properties of soft tissue by applying a mechanical vibration and measuring displacements with a motion-sensitized MRI pulse sequence [3]. The mechanical properties (e.g., the shear modulus) are calculated from the displacement field using an inverse method. Since the applied displacements are in the order of few microns, fibers may not be stretched enough to remove crimping. Therefore, it is unknown if the anisotropy of the AF due to the contribution of the fibers is detectable using MRE. The objective of this study is twofold: to measure shear properties of AF in different orientations to determine the degree of AF anisotropy observable by MRE, and to identify the contribution of different AF constituents to the measured shear modulus by applying different biochemical treatments.

The intervertebral disc plays a critical role in providing structural support to the spine while permitting extensive flexibility in a number of orientations. Axial rotation is a key parameter in spine function and torsional instability is related to spinal degeneration [1]. Animal models are integral components in many in vivo disc studies, however each animal varies in availability, size, cost, and scientific criteria such as cell phenotype and biomechanics. Selection of an appropriate animal model requires knowledge of the similarities and differences in biomechanical and biochemical factors between the model and human discs. Previous studies have often compared the characteristics of a single animal model with the human disc [2, 3]. However, variations in animal models and testing protocols between groups hinder comparisons and interpretations between different studies. This is especially relevant in torsion mechanics, where the magnitude of an applied compressive load and other testing parameters significantly affect the apparent torsional stiffness of the disc [4]. The objective of this study was to measure and compare the torsion mechanical properties of the human disc and 11 disc types from 8 mammalian species.

Fibrocartilages, including the meniscus of the knee and annulus fibrosus (AF) of the intervertebral disc, are dense connective tissues with an organized collagenous structure that play critical roles in motion and load transmission across joints. Proper mechanical function of these tissues is dependent on their structure and composition, both of which are compromised with degeneration. Tissue engineering strategies present a promising alternative to current treatments. Aligned electrospun scaffolds show promise for tissue engineering of fibrous soft tissues due to their ability to direct cell alignment and matrix deposition [1]. Furthermore, this matrix deposition results in constructs with near-native tensile properties. However, the large multi-directional forces experienced by these tissues in vivo requires that engineered constructs resist considerable shear and compressive loads as well. Unfortunately, shear properties of electrospun scaffolds are not well established. Simple shear is an appealing testing configuration because the application of tensile prestrain allows for combined fiber stretch and shear as occurs in situ. However, due to possible strain field heterogeneity, proper analysis of strain distributions must be performed [2]. The objective of this study was to quantify the effects of fiber orientation and sample aspect ratio on the shear properties of aligned electrospun scaffolds.

To compare the in vitro biomechanical properties of a 4.5 mm narrow locking compression plate (PIP-LCP) with 2 abaxially located transarticular screws and a 4.5 mm limited contact dynamic compression plate (LC-DCP) with 2 abaxially located transarticular screws using equine pasterns.Experimental. Paired in vitro biomechanical testing of 2 methods for stabilizing adult equine forelimb PIP joints.Adult equine forelimbs (n = 8 pairs).Each pair of PIP joints were randomly instrumented with either a PIP-LCP or LC-DCP plate axially and 2 parasagitally positioned 5.5 mm transarticular screws. The proximal aspect of the proximal phalanx (P1) and the distal aspect of the middle phalanx (P2) were embedded to allow for mounting on a mechanical testing machine. Each construct was tested in both cyclic and subsequently single cycle to failure in 4-point bending. The displacement required to maintain a target load of 1 kN over 3600 cycles at 1 Hz was recorded. Maximum bending moment at failure and construct stiffness was calculated from the single cycle to failure testing.In cyclic testing, significantly more displacement occurred in the LC-DCP (0.46 ± 0.10 mm) than for the PIP-LCP (0.17 ± 0.11 mm) constructs (P = .016). During single cycle testing there was no significant difference in the bending moment between the LC-DCP (148.7 ± 19.4 N m) and the PIP-LCP (164.6 ± 17.6 N m) constructs (P = .553) and the stiffness of the LC-DCP (183.9 ± 26.9 N mm) was significantly lower than for the PIP-LCP (279.8 ± 15.9 N/mm) constructs (P = .011). All constructs failed by fracture of the bone associated with the transarticular screws and subsequently bending of the plates at the middle hole.Use of the PIP-LCP resulted in a stiffer construct of the same strength as the LC-DCP in vitro using this 4-point bending model.

Introduction: Advanced Normalization Tools (ANTs) is an image registration program that has been validated for analysis of several tissues, but not previously applied to the disc. The objectives of this study were to optimize ANTs for disc image registration and to validate ANTs strain measurements by comparison with Vic2D, a commercially available software previously applied to quantify disc strain, before and after nucleotomy. Methods: Human lumbar motion segments (n=5) underwent 1000 N compression before and after nucleotomy and MR images were acquired in a reference and deformed state. Image overlap statistics were used to select mapping parameters, and strain analysis was used to select the optimal number of splines. Once the optimal registration method was selected (elastic mapping, 0.01 outlier, 6×6 splines), axial and radial strains were measured in the AAF, PAF, and IVD. Results: Excellent overlap statistics were achieved. No significant differences were found for strains calculated with ANTs and Vic2D (p≥0.35). With nucleotomy, the axial compressive strain increased in the PAF (p=0.04) and there was a trend towards decrease in radial strain (p=0.07). Discussion: ANTs is an accurate and powerful tool to calculate disc strains from MR images.

Fibrocartilages, including the knee meniscus and the annulus fibrosus (AF) of the intervertebral disc, play critical mechanical roles in load transmission across joints and their function is dependent upon well-defined structural hierarchies, organization, and composition. All, however, are compromised in the pathologic transformations associated with tissue degeneration. Tissue engineering strategies that address these key features, for example, aligned nanofibrous scaffolds seeded with mesenchymal stem cells (MSCs), represent a promising approach for the regeneration of these fibrous structures. While such engineered constructs can replicate native tissue structure and uniaxial tensile properties, the multidirectional loading encountered by these tissues in vivo necessitates that they function adequately in other loading modalities as well, including shear. As previous findings have shown that native tissue tensile and shear properties are dependent on fiber angle and sample aspect ratio, respectively, the objective of the present study was to evaluate the effects of a changing fiber angle and sample aspect ratio on the shear properties of aligned electrospun poly(e-caprolactone) (PCL) scaffolds, and to determine how extracellular matrix deposition by resident MSCs modulates the measured shear response. Results show that fiber orientation and sample aspect ratio significantly influence the response of scaffolds in shear, and that measured shear strains can be predicted by finite element models. Furthermore, acellular PCL scaffolds possessed a relatively high shear modulus, 2-4 fold greater than native tissue, independent of fiber angle and aspect ratio. It was further noted that under testing conditions that engendered significant fiber stretch, the aggregate resistance to shear was higher, indicating a role for fiber stretch in the overall shear response. Finally, with time in culture, the shear modulus of MSC laden constructs increased, suggesting that deposited ECM contributes to the construct shear properties. Collectively, these findings show that aligned electrospun PCL scaffolds are a promising tool for engineering fibrocartilage tissues, and that the shear properties of both acellular and cell-seeded formulations can match or exceed native tissue benchmarks.

The annulus fibrosus (AF) of the intervertebral disk undergoes large and multidirectional stresses and strains. Uniaxial tensile tests are limited for measuring AF material properties, because freely contracting edges can prevent fiber stretch and are not representative of in situ boundary conditions. The objectives of this study were to measure human AF biaxial tensile mechanics and to apply and validate a constitutive model to determine material properties. Biaxial tensile tests were performed on samples oriented along the circumferential-axial and the radial-axial directions. Data were fit to a structurally motivated anisotropic hyperelastic model composed of isotropic extra-fibrillar matrix, nonlinear fibers, and fiber-matrix interactions (FMI) normal to the fibers. The validated model was used to simulate shear and uniaxial tensile behavior, to investigate AF structure-function, and to quantify the effect of degeneration. The biaxial stress-strain response was described well by the model (R (2) 0.9). The model showed that the parameters for fiber nonlinearity and the normal FMI correlated with degeneration, resulting in an elongated toe-region and lower stiffness with degeneration. The model simulations in shear and uniaxial tension successfully matched previously published circumferential direction Young's modulus, provided an explanation for the low values in previously published axial direction Young's modulus, and was able to simulate shear mechanics. The normal FMI were important contributors to stress and changed with degeneration, therefore, their microstructural and compositional source should be investigated. Finally, the biaxial mechanical data and constitutive model can be incorporated into a disk finite element model to provide improved quantification of disk mechanics.

STUDY DESIGN Prospective magnetic resonance imaging (MRI) study of patients low back pain (LBP) requiring discography as part of their routine clinical diagnoses and asymptomatic age-matched volunteers. OBJECTIVE To determine whether T1ρ MRI and discography opening pressure (OP) are quantitative biomarkers of disc degeneration in LBP patients and in asymptomatic volunteers. SUMMARY OF BACKGROUND DATA Disc degenerative disease, a common cause of LBP, is related to the patient's prognosis and serves as a target for therapeutic interventions. However, there are few quantitative measures in the clinical setting. Discography OP and T1ρ MRI are potential biomarkers of disc degenerative disease related to biochemical composition of the intervertebral disc. METHODS The institutional review board approved all experiments, and informed consent was provided by each subject. Patients being treated for LBP (n = 17; 68 levels; mean age, 44 ± 6 years; and range, 30-53) and control subjects (n = 11; 44 levels; mean age, 43 ± 17 years; and range, 22-76) underwent T1ρ and T2 MRI on a Siemens 3T Tim Trio clinical scanner (Siemens Medical Solutions, Malvern, PA). The LBP patients also received multilevel provocative discography before their MRI. OP was recorded as the pressure when fluid first enters the nucleus of the intervertebral disc. RESULTS T1ρ was significantly lower in the painful discs (55.3 ± 3.0 ms, mean ± SE) from control (92.0 ± 4.9 ms, P < 0.001) and nonpainful discs (83.6 ± 3.2 ms, P < 0.001). Mean OP for the painful discs (11.8 ± 1.0 psi, mean ± SE) was significantly lower than that for nonpainful discs (19.1 ± 0.7 psi, P < 0.001). Both T1ρ and OP correlated moderately with Pfirrmann degenerative grade. Receiver-operating-characteristic area under the curve was 0.91 for T1ρ MRI and 0.84 for OP for predicting painful discs. CONCLUSION T1ρ and OP are quantitative measures of degeneration that are consistent across both control subjects and LBP patients. A significant and strong correlation exists between T1ρ values and in vivo OP measurements obtained by discography in LBP patients.

The intervertebral disc experiences combinations of compression, torsion, and bending that subject the disc substructures, particularly the annulus fibrosus (AF), to multidirectional loads and deformations. Combined tensile and shear loading is a particularly important loading paradigm, as compressive loads place the AF in circumferential hoop tension, and spine torsion or bending induces AF shear. Yet the anisotropy of AF mechanical properties in shear, as well as important structure-function mechanisms governing this response, are not well understood. The objective of this study, therefore, was to investigate the effects of tissue orientation and enzymatic degradation of glycosaminoglycan (GAG) and elastin on AF shear mechanical properties. Significant anisotropy was found: the circumferential shear modulus, Gθz, was an order of magnitude greater than the radial shear modulus, Grθ. In the circumferential direction, prestrain significantly increased the shear modulus, suggesting an important role for collagen fiber stretch in shear properties for this orientation. While not significant and highly variable, ChABC treatment increased the circumferential shear modulus compared to PBS control (p=0.15). Together with the established literature for tensile loading of fiber-reinforced GAG-rich tissues, the trends for changes in shear modulus with ChABC treatment reflect complex, structure-function relationships between GAG and collagen that potentially occur over several hierarchical scales. Elastase treatment caused no difference in shear modulus with respect to PBS control for either radial or circumferential orientation. Elastase digestion did not significantly affect shear modulus; further contributing to the notion that circumferential shear modulus is dominated by collagen fiber stretch. The results of this study highlight the complexity of the structure-function relationships that govern the mechanical response of the AF in radial and circumferential shear, and provide new and more accurate data for the validation of material models and tissue-engineered disc replacements

Understanding the interplay of composition, organization and mechanical function in load-bearing tissues is a prerequisite in the successful engineering of replacement tissues for diseased ones. Mesenchymal stem cells (MSCs) seeded on electrospun scaffolds have been successfully used to generate organized tissues that mimic fibrocartilages such as the knee meniscus and the annulus fibrosus of the intervertebral disc. While matrix deposition has been observed in parallel with improved mechanical properties, how composition, organization, and mechanical function are related is not known. Moreover, how this relationship compares to that of native fibrocartilage is unclear. Therefore, in the present work, functional fibrocartilage constructs were formed from MSC-seeded nanofibrous scaffolds, and the roles of collagen and glycosaminoglycan (GAG) in compressive and tensile properties were determined. MSCs deposited abundant collagen and GAG over 120 days of culture, and these extracellular molecules were organized in such a way that they performed similar mechanical functions to their native roles: collagen dominated the tensile response while GAG was important for compressive properties. GAG removal resulted in significant stiffening in tension. A similar stiffening response was observed when GAG was removed from native inner annulus fibrosus, suggesting an interaction between collagen fibers and their surrounding extrafibrillar matrix that is shared by both engineered and native fibrocartilages. These findings strongly support the use of electrospun scaffolds and MSCs for fibrocartilage tissue engineering, and provide insight on the structure-function relations of both engineered and native biomaterials.

Function of the annulus fibrosus (AF) of the intervertebral disc is predicated on a high degree of structural organization over multiple length scales. Recently, we have employed aligned electrospun scaffolds to engineer nanofibrous biologic laminates that replicate the form and function of the AF [1]. Further, we determined that interlamellar shearing — a direct consequence of the +/−30° angle-ply architecture — plays an important role in reinforcing the tensile response of these materials (Fig. 1). Although we have utilized fiber-reinforced continuum models to characterize the evolving mechanics of single-lamellar AF constructs with in vitro culture [2, 3], these models are not capable of capturing the interlamellar interactions observed in bi-lamellar constructs. Indeed, continuum models of the native AF typically do not account for the organization of fiber populations into discrete, alternating planes of alignment, and so these models, too, do not account for inter-lamellar shearing interactions [4–6]. Therefore, in the present work we propose a novel constitutive model for the reinforcing role of interlamellar shearing during uniaxial extension of angle-ply biologic laminates and employ this model to evaluate the functional evolution of bilayers for AF tissue engineering.Copyright © 2010 by ASME

Collagen fibers and their structural arrangement influence tissue tensile stiffness and strength. While a variety of modeling approaches incorporate collagen fibers explicitly as one of the components, due to the complexity of the fiber organization, aligned fibers are usually considered. In the pioneering work of Lanir [1], a constitutive relation for continuous fiber distributions was proposed, where the strain energy and stresses are obtained by angular integration (AI) of infinitesimal fractions of fibers aligned in a given direction. Lanir’s formulation has been successfully used to describe the mechanical behavior of a variety of tissues. In particular, Ateshian et al. [2] showed that large values of the tensile Poisson’s ratio for articular cartilage in tension and the low values observed in compression can be explained using a continuous angular distribution for the fibers. A disadvantage of the AI formulation is the large number of calculations required to evaluate the strains and stresses. On the other hand, Generalized Structure Tensors (GST) have been proposed to model tissues with continuously distributed collagen fibers [3,4]. These tensors are assumed to represent the three-dimensional distribution of the fibers. Once the tensor has been defined, the strain in the fibers can be readily obtained by multiplication with a strain tensor. The advantage of this approach is the small number of calculations required to obtain the strain energy and stresses of the fibers. As a result, this formulation can be efficiently implemented in numerical algorithms like finite elements. However, this approach is limited, as it is valid only when all of the fibers are in tension and when the fiber distribution is small [5]. A numerical comparison is required to quantify when an angular distribution can be considered acceptably small to justify using this more computationally efficient approach. The objective of this study is to numerically compare the AI and GST formulations to determine the range of values of angular distribution for which the GST approach can be accurately used.Copyright © 2010 by ASME

Mathematical modeling approaches are frequently used to characterize and predict the mechanics of biological soft tissues. Structurally-based continuum models, which describe the relationship of the constituents’ properties (i.e., collagen fibers, matrix) to overall tissue properties, require knowledge of the relationship between microscopic (fiber) and macroscopic (tissue) deformation. The most common and straightforward approach is the use of an affine model, which assumes that local fiber kinematics follow the global tissue deformation. Although the affine assumption is often used in constitutive modeling, several studies have reported non-affine fiber behavior in soft tissue testing [1–2]. Our recent work has quantified the anisotropic and inhomogeneous mechanical and organizational properties of human supraspinatus tendon (SST) [3–4]. We have also utilized a fiber dispersion model to examine SST [5]; however the relationship between macroscopic and microscopic deformation in this tendon remains unknown. Therefore, the purpose of this study was to examine the affine assumption in human SST fiber kinematics by comparing experimentally-measured fiber alignment to the affine model prediction.Copyright © 2010 by ASME

Electrospun nanofibrous scaffolds have become widely investigated for tissue engineering applications, owing to their ability to replicate the scale and organization of many fiber-reinforced soft tissues such as the knee meniscus, the annulus fibrosus of the intervertebral disc, tendon, and cartilage. However, due to their small pore size and dense packing of fibers, cellular ingress into electrospun scaffolds is limited. Progress in the application of electrospun scaffolds has therefore been hampered, as limited cell infiltration results in heterogeneous deposition of extracellular matrix and mechanical properties that remain below native benchmarks. In the present study, dynamic culture conditions dramatically improved the infiltration of mesenchymal stem cells into aligned nanofibrous scaffolds. While dynamic culture resulted in a reduction of glycosaminoglycan content, removal from dynamic culture to free-swelling conditions after 6 weeks resulted recovery of glycosaminoglycan content. Dynamic culture significantly increased collagen content, and collagen was more uniformly distributed throughout the scaffold thickness. While mechanical function was assessed and tensile modulus increased with culture duration, dynamic culture did not result in any additional improvement beyond free-swelling culture. Transient dynamic (6 weeks dynamic followed by 6 weeks free-swelling) culture significantly enhanced cell infiltration while permitting GAG accumulation. In this study, we demonstrated that a simple modification to standard in vitro culture conditions effectively improves cellular ingress into electrospun scaffolds, resolving a challenge which has until now limited the utility of these materials for various tissue engineering applications.

Study Design. To develop a construction algorithm in which electrospun nanofibrous scaffolds are coupled with a biocompatible hydrogel to engineer a mesenchymal stem cell (MSC)-based disc replacement. Objective. To engineer a disc-like angle-ply structure (DAPS) that replicates the multiscale architecture of the intervertebral disc. Summary of Background Data. Successful engineering of a replacement for the intervertebral disc requires replication of its mechanical function and anatomic form. Despite many attempts to engineer a replacement for ailing and degenerated discs, no prior study has replicated the multiscale hierarchical architecture of the native disc, and very few have assessed the mechanical function of formed neo-tissues. Methods. A new algorithm for the construction of a disc analogue was developed, using agarose to form a central nucleus pulposus (NP) and oriented electrospun nanofibrous scaffolds to form the anulus fibrosus region (AF). Bovine MSCs were seeded into both regions and biochemical, histologic, and mechanical maturation were evaluated with in vitro culture. Results. We show that mechanical testing in compression and torsion, loading methods commonly used to assess disc mechanics, reveal equilibrium and time-dependent behaviors that are qualitatively similar to native tissue, although lesser in magnitude. Further, we demonstrate that cells seeded into both AF and NP regions adopt distinct morphologies that mirror those seen in native tissue, and that, in the AF region, this ordered community of cells deposit matrix that is organized in an angle-ply configuration. Finally, constructs demonstrate functional development with long-term in vitro culture. Conclusion. These findings provide a new approach for disc tissue engineering that replicates multi-scale form and function of the intervertebral disc, providing a foundation from which to build a multi-scale, biologic, anatomically and hierarchically relevant composite disc analogue for eventual disc replacement.

Background The mechanical properties of extracellular matrix grafts that are intended to augment or replace soft tissues should be comparable to the native tissue. Such grafts are often used in fiber-reinforced tissue applications that undergo multi-axial loading and therefore knowledge of the anisotropic and nonlinear properties are needed, including the moduli and Poisson’s ratio in two orthogonal directions within the plane of the graft. The objective of this study was to measure the tensile mechanical properties of several marketed grafts: Alloderm, Restore, CuffPatch, and OrthADAPT. Methods The degree of anisotropy and non-linearity within each graft was evaluated from uniaxial tensile tests and compared to their native tissue. Findings The Alloderm graft was anisotropic in both the toe- and linear-region of the stress–strain response, was highly nonlinear, and generally had low properties. The Restore and CuffPatch grafts had similar stress–strain responses, were largely isotropic, had a linear-region modulus of 18 MPa, and were nonlinear. OrthADAPT was anisotropic in the linear-region (131 MPA vs 47 MPa in the toe-region) and was highly nonlinear. The Poisson ratio for all grafts was between 0.4 and 0.7, except for the parallel orientation of Restore which was greater than 1.0. Interpretation Having an informed understanding of how the available grafts perform mechanically will allow for better assessment by the physician for which graft to apply depending upon its application.

The highly organized structure and composition of the annulus fibrosus provides the tissue with mechanical behaviors that include anisotropy and nonlinearity. Mathematical models are necessary to interpret and elucidate the meaning of directly measured mechanical properties and to understand the structure-function relationships of the tissue components, namely, the fibers and extrafibrillar matrix. This study models the annulus fibrosus as a combination of strain energy functions describing the fibers, matrix, and their interactions. The objective was to quantify the behavior of both nondegenerate and degenerate annulus fibrosus tissue using uniaxial tensile experimental data. Mechanical testing was performed with samples oriented along the circumferential, axial, and radial directions. For samples oriented along the radial direction, the toe-region modulus was 2× stiffer with degeneration. However, no other differences in measured mechanical properties were observed with degeneration. The constitutive model fit well to samples oriented along the radial and circumferential directions (R2≥0.97). The fibers supported the highest proportion of stress for circumferential loading at 60%. There was a 70% decrease in the matrix contribution to stress from the toe-region to the linear-region of both the nondegenerate and degenerate tissue. The shear fiber-matrix interaction (FMI) contribution increased by 80% with degeneration in the linear-region. Samples oriented along the radial and axial direction behaved similarly under uniaxial tension (modulus=0.32 MPa versus 0.37 MPa), suggesting that uniaxial testing in the axial direction is not appropriate for quantifying the mechanics of a fiber reinforcement in the annulus. In conclusion, the structurally motivated nonlinear anisotropic hyperelastic constitutive model helps to further understand the effect of microstructural changes with degeneration, suggesting that remodeling in the subcomponents (i.e., the collagen fiber, matrix and FMI) may minimize the overall effects on mechanical function of the bulk material with degeneration.

Aligned nanofibrous scaffolds hold tremendous potential for the engineering of dense connective tissues. These biomimetic micropatterns direct organized cell-mediated matrix deposition and can be tuned to possess nonlinear and anisotropic mechanical properties. For these scaffolds to function in vivo, however, they must either recapitulate the full dynamic mechanical range of the native tissue upon implantation or must foster cell infiltration and matrix deposition so as to enable construct maturation to meet these criteria. In our recent studies, we noted that cell infiltration into dense aligned structures is limited but could be expedited via the inclusion of a distinct rapidly eroding sacrificial component. In the present study, we sought to further the fabrication of dynamic nanofibrous constructs by combining multiple-fiber populations, each with distinct mechanical characteristics, into a single composite nanofibrous scaffold. Toward this goal, we developed a novel method for the generation of aligned electrospun composites containing rapidly eroding (PEO), moderately degradable (PLGA and PCL/PLGA), and slowly degrading (PCL) fiber populations. We evaluated the mechanical properties of these composites upon formation and with degradation in a physiologic environment. Furthermore, we employed a hyperelastic constrained-mixture model to capture the nonlinear and time-dependent properties of these scaffolds when formed as single-fiber populations or in multipolymer composites. After validating this model, we demonstrated that by carefully selecting fiber populations with differing mechanical properties and altering the relative fraction of each, a wide range of mechanical properties (and degradation characteristics) can be achieved. This advance allows for the rational design of nanofibrous scaffolds to match native tissue properties and will significantly enhance our ability to fabricate replacements for load-bearing tissues of the musculoskeletal system.

The intervertebral disc functions over a range of dynamic loading regimes including axial loads applied across a spectrum of frequencies at varying compressive loads. Biochemical changes occurring in early degeneration, including reduced nucleus pulposus glycosaminoglycan content, may alter disc mechanical behavior and thus may contribute to the progression of degeneration. The objective of this study was to determine disc dynamic viscoelastic properties under several equilibrium loads and loading frequencies, and further, to determine how reduced nucleus glycosaminglycan content alters dynamic mechanics. We hypothesized (1) that dynamic stiffness would be elevated with increasing equilibrium load and increasing frequency, (2) that the disc would behave more elastically at higher frequencies, and finally, (3) that dynamic stiffness would be reduced at low equilibrium loads under all frequencies due to nucleus glycosaminoglycan loss. We mechanically tested control and chondroitinase-ABC injected rat lumbar motion segments at several equilibrium loads using oscillatory loading at frequencies ranging from 0.05 to 5 Hz. The rat lumbar disc behaved non-linearly with higher dynamic stiffness at elevated compressive loads irrespective of frequency. Phase angle was not affected by equilibrium load, although it decreased as frequency was increased. Reduced glycosaminoglycan decreased dynamic stiffness at low loads but not at high equilibrium loads and led to increased phase angle at all loads and frequencies. The findings of this study demonstrate the effect of equilibrium load and loading frequencies on dynamic disc mechanics and indicate possible mechanical mechanisms through which disc degeneration can progress.

Mucopolysaccharidosis VII (MPS VII) is a rare pediatric, hereditary disorder characterized by deficient activity of beta-glucuronidase, an enzyme that degrades chondroitin, dermatan and heparan sulfate glycosaminoglycans (GAGs) [1,2]. This deficiency leads to systemic lysosomal accumulation of GAGs, resulting in severely impaired physical and intellectual development, with patients frequently not surviving until adulthood. In the spine, the disease is characterized by poorly formed and aligned vertebral bodies, leading to high incidences of kyphosis and scoliosis [1–3].Copyright © 2009 by ASME

The annulus fibrosus (AF) of the intervertebral disc is a multi-lamellar fibrocartilage that, together with the nucleus pulposus, confers mechanical support and flexibility to the spine. Function of the AF is predicated on a high degree of structural organization over multiple length scales: aligned collagen fibers reside within each lamella, and the direction of alignment alternates between adjacent lamellae from +30° to −30° with respect to the transverse axis of the spine. Electrospinning permits fabrication of scaffolds consisting of aligned arrays of nanofibers, and has proven effective for directing the alignment of both cells and extracellular matrix (ECM) deposition [1–3]. We recently employed electrospinning to engineer the primary functional unit of the AF, a single lamella [4]. However, it remains a challenge to engineer a multi-lamellar tissue that replicates the cross-ply fiber architecture of the native AF. Moreover, relatively few studies have considered functional properties of engineered AF, and, when measured, tensile properties of these constructs have been inferior to native AF [4]. In this study, mesenchymal stem cells (MSCs) were seeded onto aligned nanofibrous scaffolds organized into bi-lamellar constructs with opposing or parallel fiber orientations, and their functional maturation was evaluated with time. Additionally, we determined a novel role for inter-lamellar ECM in reinforcing the tensile response of bilayers, and confirmed this mechanism by testing acellular bilayers with controllable interface properties.Copyright © 2009 by ASME

The annulus fibrosus (AF) is a highly organized structure made up of concentric lamellae of fibers embedded in a hydrated extrafibrillar matrix; the collagen fibers are oriented at alternating angles in each lamella. The AF undergoes multidirectional loading through combinations of compression, bending, torsion and shear of the motion segment. The composition and structure of the AF leads to mechanical stress-strain nonlinearity and anisotropy. Previous tissue-based studies of shear have tested the AF tissue under compressive simple shear and torsion, producing shear modulus on the order of 0.06–0.4 MPa [1, 2]. However, structural testing and mathematical models of the IVD have reported the shear modulus to be between 3–20 MPa [3–6]. We hypothesize that when the fibers of the AF are loaded the shear modulus will be on the same order as structural tests and mathematical models of the IVD. The objectives of this study are to measure the shear mechanical properties of the bovine outer AF and compare the regional variances between anterior and posterior AF.

Rotator cuff tears may be due in part to the complex loading environment of the supraspinatus tendon (SST). Previous research has reported inhomogeneous uniaxial tensile mechanical properties of human SST [1–2] and location-specific collagen fiber alignment distributions that are qualitatively more disperse than other tendons [3–4]. Our group recently measured fiber alignment under load of samples tested along the tendon long-axis and found that re-alignment occurs in the toe-region and varies by SST location [5]. However, the mechanical properties and effect of fiber alignment under more complex loads remain unknown. Examining the properties of SST when tested transverse to the tendon long-axis will evaluate tissue anisotropy and better elucidate possible mechanisms for tissue inhomogeneity and nonlinearity. Therefore, the objectives of this study are to 1) measure local fiber alignment during transverse tensile loading, 2) measure corresponding mechanical properties, and 3) examine structure-function relationships of SST. We hypothesize that 1) fibers will become less aligned during transverse testing, 2) mechanical properties will be greatest in the anterior and bursal locations, and 3) higher initial alignment will correspond to lower transverse tensile properties.Copyright © 2009 by ASME

Tendon tissue is composed of collagen fibers in a hydrated proteoglycan matrix. Although many tendons have fibers that are highly aligned (e.g. flexor tendon), the supraspinatus tendon (SST) of the shoulder has significant distribution of fiber alignment [1]. The alignment and distribution of the fibers likely contributes to the nonlinear and anisotropic mechanical behavior, however this has not been demonstrated. Understanding the role of fiber structure on tendon mechanical behavior, that is, characterizing the structure-function relationships, is critical to evaluate the function of injured, degenerated, or healing tendons and would be invaluable in the design and assessment of tissue engineered tendon replacements. While a structurally based hyperelastic model has been developed for tendon [2], this model contained only a single fiber orientation, which is not adequate for the more distributed fiber structure in the SST. We have recently applied a hyperelastic model formulation that has distributed collagen fiber orientation developed by Gasser and colleagues for the arterial wall [3] to model a tendon analog made from nanofibrous scaffolds [4]. The objective of this study was to build on previous work to apply a hyperelastic fiber-reinforced constitutive model that includes a specific term for fiber distribution to the tensile mechanics of human SST and to evaluate the site-specific model material properties.

A recent study (Lake et al. 2009) reported the properties of human supraspinatus tendon (SST) tested along the predominant fiber direction. The SST was found to have a relatively disperse distribution of collagen fibers, which may represent an adaptation to multiaxial loads imposed by the complex loading environment of the rotator cuff. However, the multiaxial mechanical properties of human SST remain unknown. The objective of this study, therefore, was to evaluate the mechanical properties, fiber alignment, change in alignment with applied load, and structure-function relationships of SST in transverse testing. Samples from six SST locations were tested in uniaxial tension with samples oriented transverse to the tendon long-axis. Polarized light imaging was used to quantify collagen fiber alignment and change in alignment under applied load. The mechanical properties of samples taken near the tendon-bone insertion were much greater on the bursal surface compared to the joint surface (e.g., bursal moduli 15–30 times greater than joint; p

Reduced nucleus pulposus glycosaminoglycan (GAG) content is one of the earliest clinically detectable changes during the course of intervertebral disc degeneration [1,2]. Depletion of nucleus GAG by small percentages consistent with this early loss has been experimentally linked to altered motion segment mechanical function, and thus, potentially increases the risk of damage accumulation directly due to elevated stresses and strains and through altered cellular function [3]. Recently, our laboratory has established an in vivo model in a rat lumbar disc which moderately decreases nucleus GAG to levels observed in early human degeneration. In this model, GAG loss is accompanied by a state of hypermobility at both 4 and 12 weeks post treatment [4], potentially making the disc susceptible to mechanical failure. The objective of this study was to determine the long term effects of nucleus GAG depletion and to determine if altered discs demonstrate hallmark features of disc degeneration. We hypothesized that GAG will remain depleted 24 weeks post treatment, potentially decreasing to lower levels, and further that geometrical and mechanical changes consistent with degeneration will be observed.Copyright © 2008 by ASME

Physiological cyclic loading of the intervertebral disc generally occurs between 1 and 5 Hz [1]. However, most mechanical assessments of disc AF tissue have relied on uniaxial tensile tests under quasi-static conditions [2]. Furthermore, the few studies that have addressed AF viscoelasticity have reported only static viscoelastic properties such as creep or stress-relaxation [3]. As such, there exists almost no data characterizing the dynamic viscoelastic properties of AF tissue in tension. Such data would be critical for several applications: to elucidate the mechanical progression of intervertebral disc degeneration, to develop and validate structural finite element models, and to provide native tissue benchmarks for regenerative approaches that aim to restore mechanical function to diseased or degenerate tissue. Therefore, the objectives of this study are to: (1) quantify human AF tissue frequency-dependent and strain-dependent viscoelastic properties of human AF tissue in circumferential tension, and (2) determine the effects of disc degeneration on these properties.Copyright © 2008 by ASME

The intervertebral disc is comprised of complex components that provide the disc with nonlinear, viscoelastic and anisotropic mechanical properties. The annulus fibrosus (AF) is a highly organized structure composed of concentric layers of collagen fibers embedded in a proteoglycan matrix. The AF has a high tensile stiffness and supports the large loads encountered by the disc. Mathematical models are needed to interpret and elucidate the meaning of experimental measurements made in mechanical tests. Based upon the classic work of Spencer [1], the AF has been modeled as a fiber-induced anisotropic hyperelastic material [e.g.,2–6], using the principle invariants of the Green deformation tensor and structural tensors representing the collagen fiber populations. Contributions of other AF components to mechanical behaviors are less understood than the fibers or matrix and may include connections between collagens and proteoglycans that can be incorporated into models through fiber-matrix interactions [2–4]. The previous models, however, have not been applied to experimental data from both nondegenerate and degenerate tissue. Constitutive modeling applied to nondegenerate and degenerate AF may elucidate microstructural changes with degeneration, will be useful for finite element models [5], and provide targets for disc treatments, such as tissue engineered constructs [7].Copyright © 2008 by ASME

The intervertebral disc has the important biomechanical function of dissipating energy during spinal loading. With degeneration, the disc experiences, among other changes, a loss of mechanical function and degradation of its composition. Using a rat model of early disc degeneration by injection of Chondroitinase ABC (ChABC), glycosaminoglycan (GAG) content in the nucleus pulposus (NP) was reduced which altered neutral zone mechanical properties. The contribution of decreased NP GAG content to the dynamic viscoelastic properties has yet to be determined. The advantage of dynamic viscoelastic testing is that it provides both viscous and elastic stiffness values as a function of loading frequency. These methods have been employed previously in a rabbit disc regeneration model, in ligament under three modes of loading, and in NP under oscillatory shear and compression. Therefore, the objective of this study was to determine the viscoelastic behavior of a rat lumbar disc at several equilibrium strains and to quantify the impact of GAG reduction on this behavior. Our hypotheses were: 1) elastic stiffness would be greater, and viscous stiffness and loss angle would be lower with increased frequency, and 2) both elastic and viscous stiffness would be lower in the reduced GAG discs at all frequencies.Copyright © 2008 by ASME

Study design Internal deformations and strains were measured within intact human motion segments. Objective Quantify 2-dimensional internal deformation and strain in compression of human intervertebral discs using MRI. Summary of background data Experiments using radiographic or optical imaging have provided important data for internal disc deformations. However, these studies are limited by physical markers and/or disruption of the disc structural integrity. Methods MR images were acquired before and during application of a 1000 N axial compression. Two-dimensional internal displacements, average strains, and the location and direction of peak strains were calculated using texture correlation, a pattern matching algorithm. Results The average height loss was 0.4 mm, which corresponded to 4.4% compressive strain. The inner AF radial displacement was outward, even with degeneration; the average outward displacement of the inner AF (0.16 mm) was less than the outer AF (0.36 mm). High shear peak strains (2%-26%) occurred near the endplate and at the inner AF. Shear was higher in the anterior AF compared to the posterior. Conclusion This technique allows quantification of displacement and strain within the intact disc. The radial displacements of inner AF suggest NP translation under compression. Peak tensile radial strains occurred as vertical bands throughout the anulus, which may contribute to radial tears and herniations. The tensile axial and shear strains at the interface between the AF and endplate could be related to the occurrence of rim lesions. Peak strains at the endplate are likely due to the AF curvature and the oblique fibers angle at fiber insertion sites. In the future, this technique may be used to measure disc strain under a variety of loading conditions, such as bending or torsion, and could also be used to study the mechanical effects of disc degeneration and potential clinical interventions.

Intervertebral disc degeneration is a complex progression of structural, biochemical, and biological alterations that contribute to compromised mechanical function and in some instances discogenic low back pain. Despite the prevalence of the condition, with billions of dollars in associated health care costs paid annually in the United States,1 treatments for disc degeneration and the associated pain are limited, due in part to the lack of a thorough understanding of the mechanisms at play. Among the early degenerative changes is a breakdown of large aggregating proteoglycans reducing the sulfated glycosaminoglycan content in the nucleus pulposus.2–4 This reduction of glycosaminoglycan content has an impact on the mechanical function of the disc: the ability to imbibe and bind water is diminished, pressure within the nucleus is decreased, and ultimately, the mechanical function of the nucleus and the entire motion segment is altered.5–8 It is plausible that progressive degeneration follows this reduced glycosaminoglycan content and altered mechanics, yet this ultimately remains a hypothesis, and knowledge of specific mechanisms and interactions is limited. To this end, in vivo animal models simulating the condition of decreased nucleus pulposus glycosaminoglycan content as observed in early degeneration would be of great utility. Such models would allow the study of the links between a relevant altered biochemistry, altered mechanical function, and altered cellular function, further allowing for the development of specific therapies targeted at bolstering anabolic and inhibiting catabolic cellular function, ultimately restoring disc structure and mechanical function. The experimental reduction of the glycosaminoglycan content in the nucleus is not a new concept; however, to date this has not been performed in a controlled setting where a reduction of a physiologically relevant quantity of glycosaminoglycan has been achieved as an isolated factor. Chemonucleolysis studies using the enzyme chymopapain date back over 40 years, and were developed as a treatment for disc herniations through a reduction in nucleus pulposus pressure.9 Negative outcomes from chymopapain led to the later introduction of chondroitinase ABC (ChABC) as a herniation treatment as this enzyme selectively degrades chondroitin-4 sulfate, chondroitin-6 sulfate, and dermatan sulfate glycosaminoglycan chains10,11 and is less aggressive than chymopapain. More recently, after the observations of a degeneration-like loss of pressure and disc height,12,13 ChABC has been specifically used to induce degenerative changes using in vivo animal models.14,14a Common limitations in previous ChABC injection studies include the utilization of excessive quantities of enzyme sufficient to render discs devoid of glycosaminoglycan chains and the use of large needle diameters relative to overall disc heights, which has been shown to induce degenerative changes.15–17a The objective of the current study was to establish an in vivo model in the rat lumbar spine simulating the diminished glycosaminoglycan and altered mechanics observed in early disc degeneration using a controlled injection of ChABC via a sufficiently small needle. The rat lumbar intervertebral disc was selected for this model, as this disc has shown many similarities to human lumbar discs in terms of biomechanics, biochemistry, cellularity, and geometry.18–21 The rat also has the advantage over other larger models by providing a relatively inexpensive platform for the future implementation of many previously established biologic and genetic assays and treatments. Moreover, we have previously shown in vitro that an injection with a 33-gauge needle does not cause alterations in mechanical function in the rat lumbar disc, and that glycosaminoglycan can be reduced to a physiologic level through ChABC injection.5 For the present study, we hypothesized that the injection procedure itself would not alter disc properties, and further, that changes in disc height, mechanics, biochemistry, and histologic structure consistent with human degeneration would be observed due to glycosaminoglycan degradation. Additionally, we hypothesized that degeneration-like changes would be evident at 4 weeks after glycosaminoglycan reduction and that these changes would progress at 12 weeks. Specifically, we hypothesized that glycosaminoglycan reduction and the altered biochemical profile would result in progressively decreased disc height, hypermobility (increased axial range of motion and decreased neutral zone modulus), and increased viscoelastic creep strain.

Study design Quantitative study of elastin content in nondegenerate and degenerate human intervertebral discs. Objective To measure the site-specific changes in elastin content that accompany disc degeneration using a quantitative, dye-binding assay to assess elastin levels. Summary of background data Recently, an abundant and organized network of elastic fibers was observed in nondegenerated human disc using immunostaining histochemistry, suggesting a functional role for elastin. While degenerative changes in the disc extracellular matrix composition are well known, changes in elastin content that may accompany degeneration have not been reported. Methods Human discs were assigned a degenerative grade by 3 independent orthopedic surgeons based on gross morphology. Samples were taken from the outer anulus fibrosus (OAF), inner AF (IAF) and nucleus pulposus (NP). Elastin content was measured using a specific, dye-binding assay and normalized to dry weight and collagen content, which was measured via a hydroxyproline assay. Samples were divided into 2 groups: nondegenerate (Grades 1-2.5) and degenerate (Grades 2.6-4.0). A 2-way analysis of variance was used to test for statistical significance where the 2 factors were disc location and degeneration. Correlations of composition with degeneration and age were analyzed. Results In nondegenerate tissue, elastin by dry weight was on average 2.0% +/- 0.3%, and there were no differences in elastin content among the locations of OAF, IAF, or NP. With degeneration, there was a significant increase in total disc elastin per dry weight at each location. The degenerate IAF had the largest amount of elastin (9.3% +/- 2.3%), significantly greater than the NP and OAF. Elastin content correlated with degenerative grade and age at each site. Conclusion Based on the location-dependent degenerative changes, with highest increases in the IAF, elastin may function to restore lamellar structure under radial loads that potentially cause delamination. Future work will focus on distinguishing the changes in elastin orientation with degeneration and understanding the mechanical functional role of elastin in the disc.

Endplate subsidence and vertebral body (VB) fracture are potential complications following lumbar total disc replacement (TDR) [1]. Early clinical evidence suggests that these events can be ameliorated in patients with osteopenia and osteoporosis by vertebral augmentation performed at the time of TDR [2]. However, the biomechanical basis to support vertebral augmentation of TDR has not been established. The objective of this study was to quantify the effects of vertebral augmentation with Cortoss on VB mechanics under compression by a TDR implant. We hypothesize that augmentation with Cortoss will improve the mechanical behavior in compression.Copyright © 2007 by ASME