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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

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.

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.

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.

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.

Loss of or damage to the meniscus alters the pattern of loading in the knee joint and frequently leads to cartilage degeneration and osteoarthritis. The mechanical properties of articular cartilage have been shown to reflect the extent of cartilage degeneration in human osteoarthritis and in experimental models of joint disease, but there is little experimental data documenting changes in cartilage mechanics following meniscectomy. We hypothesized that the tensile properties of the surface zone of articular cartilage are altered following total medial meniscectomy. Twelve mongrel dogs underwent complete resection of the medial meniscus in the right knee, and the femoral cartilage was studied 12 weeks after the operation. We performed uniaxial, tensile stress-relaxation tests to determine the equilibrium tensile modulus of surface-zone cartilage. Water and glycosaminoglycan content were also measured at site-matched locations. The tensile moduli of the cartilage decreased significantly following meniscectomy. The linear region modulus decreased by 40%, from 25.5 +/- 7.7 to 15.3 +/- 7.2 MPa. There was a weak (r = -0.45), but significant, correlation between the linear region modulus and the gross morphological grade for cartilage damage. Water and glycosaminoglycan content did not change following meniscectomy. Composition was not correlated with mechanical properties or morphological grade, suggesting that cartilage structure may play a more important role than composition in determining the mechanical properties. The observed decrease in cartilage material properties provides a quantitative measure of the loss of cartilage function following meniscectomy and reflects a pattern of change that is consistent with damage to the collagen-proteoglycan solid network.